Microfluidic particle-analysis systems

ABSTRACT

The invention provides systems, including apparatus, methods, and kits, for the microfluidic manipulation and/or detection of particles, such as cells and/or beads. The invention provides systems, including apparatus, methods, and kits, for the microfluidic manipulation and/or analysis of particles, such as cells, viruses, organelles, beads, and/or vesicles. The invention also provides microfluidic mechanisms for carrying out these manipulations and analyses. These mechanisms may enable controlled input, movement/positioning, retention/localization, treatment, measurement, release, and/or output of particles. Furthermore, these mechanisms may be combined in any suitable order and/or employed for any suitable number of times within a system. Accordingly, these combinations may allow particles to be sorted, cultured, mixed, treated, and/or assayed, among others, as single particles, mixed groups of particles, arrays of particles, heterogeneous particle sets, and/or homogeneous particle sets, among others, in series and/or in parallel. In addition, these combinations may enable microfluidic systems to be reused. Furthermore, these combinations may allow the response of particles to treatment to be measured on a shorter time scale than was previously possible. Therefore, systems of the invention may allow a broad range of cell and particle assays, such as drug screens, cell characterizations, research studies, and/or clinical analyses, among others, to be scaled down to microfluidic size. Such scaled-down assays may use less sample and reagent, may be less labor intensive, and/or may be more informative than comparable macrofluidic assays.

CLAIM OF PRIORITY

[0001] This application claims the benefit of priority under 35 U.S.C.§119(e) to provisional applications Serial No. 60/369,538, filed Apr. 1,2002 and 60/378,464, filed May 6, 2002, both of which are herebyincorporated by reference in their entirety for all purposes and thosepurposes stated herein and therein. This application further claimspriority under 35 U.S.C. §120 as a continuation-in-part of thenon-provisional patent application titled “MicrofluidicParticle-Analysis Systems”, by Chou et al., filed on Mar. 31, 2003(Atty. Docket No.: 139F.310US), which is hereby incorporated byreference for all purposes.

CROSS-REFERENCES TO PATENT APPLICATIONS

[0002] This application incorporates by reference in their entirety forall purposes the following U.S. patent applications: Ser. No.09/605,520, filed Jun. 27, 2000; Ser. No. 09/724,784, filed Nov. 28,2000; Ser. No. 09/724,967, filed Nov. 28, 2000; Ser. No. 09/796,378,filed Feb. 28, 2001; Ser. No. 09/796,666, filed Feb. 28, 2001; Ser. No.09/796,871, filed Feb. 28, 2001; Ser. No. 09/826,583, filed Apr. 6,2001; and Ser. No. 09/724,784, filed Nov. 28, 2001, titledMICROFABRICATED ELASTOMERIC VALVE AND PUMP SYSTEMS, and naming Marc A.Unger, Hou-Pu Chou, Todd A. Thorsen, Axel Scherer, Stephen R. Quake,Jian Liu, Mark L. Adams, and Carl L. Hansen as inventors.

CROSS-REFERENCES TO OTHER MATERIALS

[0003] This application incorporates by reference in their entirety forall purposes the following publications: Joe Sambrook and David Russell,Molecular Cloning: A Laboratory Manual (3^(rd) ed. 2000); and R. IanFreshney, Culture of Animal Cells: A Manual of Basic Technique (4^(th)ed. 2000).

FIELD OF THE INVENTION

[0004] The invention relates to systems for the manipulation and/ordetection of particles. More particularly, the invention relates tomicrofluidic systems for the manipulation and/or detection of particles,such as cells and/or beads.

BACKGROUND OF THE INVENTION

[0005] The ability to perform molecular and cellular analyses ofbiological systems has grown explosively over the past three decades. Inparticular, the advent and refinement of molecular and cellulartechniques, such as DNA sequencing, gene cloning, monoclonal antibodyproduction, cell transfection, amplification techniques (such as PCR),and transgenic animal formation, have fueled this explosive growth.These techniques have spawned an overwhelming number of identifiedgenes, encoded proteins, engineered cell types, and assays for studyingthese genes, proteins, and cell types. As the number of possiblecombinations of samples, reagents, and assays becomes nearlyincalculable, it has become increasingly apparent that novel approachesare necessary even to begin to make sense of this complexity, especiallywithin reasonable temporal and monetary limitations.

[0006] One approach to these difficulties has been to reduce the scaleof assays. Accordingly, substantial effort has been directed todeveloping assay methods and instrumentation for high-density microtiterplates. However, very small assay volumes in high-density microtiterplates, particularly assays with cells, may suffer from a number ofshortcomings. For example, cells may be lost easily from wells, may beharmed by rapid fluid evaporation, may contaminate nearby wells, and maybe difficult to remove efficiently from wells for additional analysis orculture. Thus, there is a need for systems that can effectivelymanipulate and analyze cells and other small particles, such as beads,in small volumes.

SUMMARY OF THE INVENTION

[0007] The invention provides systems, including apparatus, methods, andkits, for the microfluidic manipulation and/or detection of particles,such as cells and/or beads.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 is a flow chart showing potential temporal relationshipsbetween method steps for manipulation and/or detection of particles in amicrofluidic system, in accordance with aspects of the invention.

[0009]FIG. 2A is a top plan view of a microfluidic system for retainingand analyzing a subset of input particles, in accordance with aspects ofthe invention.

[0010]FIG. 2B is a top plan view of another microfluidic system forretaining and analyzing a subset of input particles, in accordance withaspects of the invention.

[0011]FIG. 3 is a fragmentary, top plan view of yet another microfluidicsystem for retaining and analyzing a subset of input particles, inaccordance with aspects of the invention.

[0012]FIG. 4 is a view of the system of FIG. 3 during particlepositioning and retention, illustrating the various flow paths followedby particles, in accordance with aspects of the invention.

[0013]FIG. 5 is a fragmentary, top plan view of a microfluidic systemfor positioning and retaining a group of particles, and for perfusingthe retained group with selected reagents, in accordance with aspects ofthe invention.

[0014]FIG. 6 is a photographic image of a portion of a chip fabricatedaccording to the system of FIG. 5, in accordance with aspects of theinvention.

[0015]FIG. 7 is a schematic rendition of the image of FIG. 6,illustrating paths of fluid flow and particle movement relative to aparticle-retention or capture chamber, in accordance with aspects of theinvention.

[0016]FIG. 8 is a full top plan view of the system of FIG. 5.

[0017]FIG. 9 is a photographic image of cells in a retention chamber,after exposure to Trypan blue to stain lysed cells, but before cellfixation, in accordance with aspects of the invention.

[0018]FIG. 10 is another photographic image of the cells and chamber ofFIG. 9, after exposure to methanol to lyse and fix the cells, inaccordance with aspects of the invention.

[0019]FIG. 11 is yet another photographic image of the cells and chamberof FIG. 9, after exposure to 1) methanol to lyse and fix the cells, 2)Trypan blue to stain lysed cells, and 3) a wash buffer to remove excessTrypan blue, in accordance with aspects of the invention.

[0020]FIG. 11A is a fragmentary, top plan view of a microfluidic systemfor measuring cell-cell communication, based on a duplicated version ofthe system of FIG. 8, in accordance with aspects of the invention.

[0021]FIG. 11B is a top plan view of selected portions of an alternativeembodiment of the system of FIG. 11A, in accordance with aspects of theinvention.

[0022]FIG. 11C is a top plan view of a two-dimensional array of particlecapture chambers that may be used in a microfluidic system, inaccordance with aspects of the invention.

[0023]FIG. 12 is a fragmentary, top plan view of a microfluidic systemfor retaining and perfusing two sets of particles in parallel, inaccordance with aspects of the invention.

[0024]FIG. 13 is a view of selected portions of the system of FIG. 12,illustrating paths for fluid flow and particle movement relative to twoadjacent retention chambers, in accordance with aspects of theinvention.

[0025]FIG. 13A is a top plan view of a microfluidic system for retainingtwo particles at spaced sites in a channel and perfusing the retainedparticles with distinct reagents, in accordance with aspects of theinvention.

[0026]FIG. 13B is a top plan view of selected portions of the system ofFIG. 13A, in accordance with aspects of the invention.

[0027]FIG. 13C is a top plan view of selected portions of an alternativeembodiment of the system of FIG. 13A, in accordance with aspects of theinvention.

[0028]FIG. 13D is a photograph of two beads being exposed to green dyedelivered by spaced treatment mechanisms, using a chip constructedaccording to the system of FIG. 13A, in accordance with aspects of theinvention.

[0029]FIG. 13E is another photograph of the two beads of FIG. 13D duringexposure to a red dye and a green dye delivered by spaced treatmentmechanisms, in accordance with aspects of the invention.

[0030]FIG. 13F is yet another photograph of the two beads of FIG. 13Dduring exposure to a red dye and a yellow dye delivered by spacedtreatment mechanisms, in accordance with aspects of the invention.

[0031]FIG. 13G is a photograph of two cells held at separate retentionsites in a chip constructed according to the system of FIG. 13A, inaccordance with aspects of the invention.

[0032]FIG. 13H is a photograph of the two cells of FIG. 13G duringexposure to a blue dye delivered by spaced treatment mechanisms, inaccordance with aspects of the invention.

[0033]FIG. 13I is a photograph of the two cells of FIG. 13G duringtreatment of only one of the cells with an organic fixative, inaccordance with aspects of the invention.

[0034]FIG. 13J is a photograph of the two cells of FIG. 131, afterfixation of the one cell and during exposure to a blue dye, delivered byspaced treatment mechanisms, in accordance with aspects of theinvention.

[0035]FIG. 13K is a photograph of two fluorescent beads held at tworetention sites and individually exposed to a fluorescent and achromophoric dye delivered by spaced treatment mechanisms, but withoutthe use of a spacer buffer, using a chip constructed according to thesystem of FIG. 13A, in accordance with aspects of the invention.

[0036]FIG. 13L is a fragmentary, top plan view of a microfluidic systemhaving separately addressable sets of linear trap arrays, in accordancewith aspects of the invention.

[0037]FIG. 14 is a top plan view of a microfluidic system for retainingan array of particles in series and for perfusing members of this arrayseparately and in parallel, in accordance with aspects of the invention.

[0038]FIG. 15 is a top plan view of selected portions of the system ofFIG. 14, illustrating fluid-layer and control-layer networks fortreating retained particles separately and in parallel, in accordancewith aspects of the invention.

[0039]FIG. 16 is a top plan view of portions of a single retentionnetwork from the system of FIG. 14, illustrating selected paths of fluidflow, in accordance with aspects of the invention.

[0040]FIG. 17 is a fragmentary, top plan view of a microfluidic devicefor forming an array of single particles or groups of particles, inaccordance with aspects of the invention.

[0041]FIG. 18 is a pair of fragmentary, top plan schematic views of amicrofluidic device for forming an array of retained particles that maybe transferred to an array of separate sites, illustrating particleretention and transfer configurations, on the left and rightrespectively, in accordance with aspects of the invention.

[0042]FIG. 19 is a pair of fragmentary, top plan schematic views ofanother microfluidic device for forming an array of retained particlesthat may be transferred to an array of separate sites, illustratingparticle retention and transfer configurations, on the left and rightrespectively, in accordance with aspects of the invention.

[0043]FIG. 20 is fragmentary, top plan schematic view of yet anothermicrofluidic device for forming an array of retained particles that maybe transferred to an array of separate sites, in accordance with aspectsof the invention.

[0044]FIG. 21 is a composite of top plan and sectional views showingselected portions of a microfluidic system for retaining particles usinga particle-retention chamber that is fully spaced from the floor of thesystem, in accordance with aspects of the invention.

[0045]FIG. 22 is a composite of top plan and sectional views, and aphotographic image, showing selected portions of a microfluidic systemfor retaining particles using a particle-retention chamber that ispartially spaced from the floor of the system, in accordance withaspects of the invention.

[0046]FIG. 23 is a composite of top plan and sectional views, and twophotographic images, showing selected portions of another microfluidicsystem for retaining particles using a particle-retention chamber thatis fully spaced from the floor of the system, in accordance with aspectsof the invention.

[0047]FIG. 24 is a fragmentary, top plan view of a reusable microfluidicsystem for repeated retention, treatment, and release of singleparticles, in accordance with aspects of the invention.

[0048]FIG. 25 is a view of selected portions of the system of FIG. 24,particularly a particle release mechanism, in accordance with aspects ofthe invention.

[0049]FIG. 26 is a fragmentary, top plan view of a reusable microfluidicsystem for repeated retention, treatment, and release of groups ofparticles, in accordance with aspects of the invention.

[0050]FIG. 27 is a view of selected portions of the systems of FIGS. 24and 26, particularly a particle collection mechanism, in accordance withaspects of the invention.

[0051]FIG. 28 is a fragmentary, top plan view of an input mechanism thatincludes a particle suspension mechanism, in accordance with aspects ofthe invention.

[0052]FIG. 29 is a fragmentary, top plan view of an adjustable dilutionmechanism, in accordance with aspects of the invention.

[0053]FIG. 30 is a fragmentary, top plan view of another adjustabledilution mechanism, in accordance with aspects of the invention.

[0054]FIG. 31 is a top plan view of a microfluidic system having asorting mechanism based on centrifugal force, in accordance with aspectsof the invention.

[0055]FIG. 32 is a fragmentary view of the system of FIG. 31, showingthe sorting mechanism in greater detail, in accordance with aspects ofthe invention.

[0056]FIG. 33 is a fragmentary, top plan view of another microfluidicsystem having a sorting mechanism based on centrifugal force, inaccordance with aspects of the invention.

[0057]FIG. 34 is a top plan view of a yet another microfluidic systemhaving a sorting mechanism based on centrifugal force, in accordancewith aspects of the invention.

[0058]FIG. 35 is a fragmentary view of the system of FIG. 34, showingthe sorting mechanism in greater detail.

[0059]FIG. 36 is a photographic image of fluorescent beads and particlesbeing separated by the sorting mechanism of FIGS. 34 and 35.

[0060]FIG. 37 is a graph plotting the ratio of cells to beads over timeduring sorting with the system of FIGS. 34 and 35.

[0061]FIG. 38 is a graph plotting the ratio of cells to beads over timeduring sorting with the system of FIGS. 31 and 32.

[0062] FIGS. 39-43 are top plan composite views of various cell-chambernetworks for microfluidic manipulation of cells, in accordance withaspects of the invention.

[0063]FIG. 44 is a top plan view of a microfluidic system with aparallel array of separate, isolatable cell-chamber networks, inaccordance with aspects of the invention.

[0064]FIG. 45 is a top plan view of a microfluidic system with anisolatable cell chamber that may be fed or bypassed by a parallelfluidic circuit, in accordance with aspects of the invention.

[0065]FIG. 46 is a top plan view of a microfluidic system having a cellchamber that forms a loop, in accordance with aspects of the invention

[0066]FIG. 47 is a top plan view of a microfluidic system in whichparticle and reagent networks intersect at a common cell chamber, inaccordance with aspects of the invention.

[0067]FIGS. 48 and 49 are photographic images of filtering mechanismswith size-selective channels that are included in the reagent networksof chips fabricated according to the system of FIG. 47.

[0068]FIG. 50 is a composite of two photographic images showing cellscultured in a cell chamber of a chip fabricated according to the systemof FIG. 47.

[0069]FIG. 50A is a fragmentary, top plan view of a system fordepositing cells in a cell chamber, based on a nonlinear, asymmetricalflow path, in accordance with aspects of the invention.

[0070]FIG. 50B is a fragmentary, top plan view of a modified version ofthe system of FIG. 50A, in which reagent(s) may be recirculated throughthe cell chamber, in accordance with aspects of the invention.

[0071]FIG. 50C is a top plan view of a cell chamber having two distinctcompartments connected by a set of radially arrayed, size-selectivechannels, in accordance with aspects of the invention.

[0072]FIG. 50D is a top plan view of a version of the cell chamber ofFIG. 50C, modified to interconnect the two compartments more fully, inaccordance with aspects of the invention.

[0073]FIG. 51 is an isometric schematic view of a microfluidic systemfor performing electrophysiological analysis on an array of cells, inaccordance with aspects of the invention.

[0074]FIG. 52 is a top plan view of a microfluidic system for performingelectrophysiological analysis on a single cell, in accordance withaspects of the invention.

[0075]FIG. 53 is a fragmentary top plan view of a microfluidic systemrelated to the system of FIG. 52, showing a modified focusing mechanism,in accordance with aspects of the invention.

[0076]FIG. 54 is a top plan view of selected portions of the system ofFIG. 52 with a retained cell, in accordance with aspects of theinvention.

[0077]FIG. 55 is a top plan view of selected portions of the system ofFIG. 52 during perfusion of a retained cell, in accordance with aspectsof the invention.

[0078]FIG. 56 is another top plan view of selected portions of thesystem of FIG. 52, in accordance with aspects of the invention.

[0079]FIG. 57 is yet another top plan view of selected portions of thesystem of FIG. 52, in accordance with aspects of the invention.

[0080]FIG. 58 is a photographic image of a portion of a chip fabricatedaccording to the system of FIG. 52.

[0081]FIG. 59 is an abstracted view of a microfluidic device forperforming patch-clamp analysis of cells, in accordance with aspects ofthe invention.

[0082]FIG. 60 is a fragmentary top plan view of a microfluidic devicefor performing patch-clamp analysis of multiple individual cells, inaccordance with aspects of the invention.

[0083]FIG. 61 is a graph showing 95% probability of successfullyobtaining an electrophysiological reading as a function of both thenumber of apertures (channels) analyzed and the fraction of individualapertures that give a successful reading.

[0084]FIG. 62 is a fragmentary side elevation view of a microfluidicmold spin-coated with a first layer of patternable, selectivelyremovable material, in accordance with aspects of the invention.

[0085]FIG. 63 is a fragmentary side elevation view of the mold of FIG.62 after patterned removal of the first layer, in accordance withaspects of the invention.

[0086]FIG. 64 is a fragmentary side elevation view of the mold of FIG.63 spin-coated with a second layer of patternable, selectively removablematerial, in accordance with aspects of the invention.

[0087]FIG. 65 is a fragmentary side elevation view of the mold of FIG.64 after patterned removal of the second layer, in accordance withaspects of the invention.

[0088]FIG. 66 is a fragmentary side elevation view of the mold of FIG.65 after heating at elevated temperatures to round remaining portions ofthe second layer, in accordance with aspects of the invention.

[0089]FIG. 67 is a fragmentary side elevation view of the mold of FIG.66 spin-coated with a third layer of patternable, selectively removablematerial, in accordance with aspects of the invention.

[0090]FIG. 68 is a fragmentary side elevation view of the mold of FIG.67 following patterned removal of the third layer, in accordance withaspects of the invention.

[0091]FIG. 69 is a fragmentary side elevation view of the mold of FIG.68 acting to mold complementary surface features of a fluid-layermembrane, in accordance with aspects of the invention.

[0092]FIG. 70 is a composite of photographic images of 1) a fluid-layermold formed using the method depicted in FIGS. 62-68 and 2) acorresponding molded chip formed from the fluid-layer mold, inaccordance with aspects of the invention.

[0093]FIG. 71 is a composite of photographic images of 1) a fluid-layermold formed using the method depicted in FIGS. 62-68 and 2) acorresponding molded chip formed partially from the fluid-layer mold, inaccordance with aspects of the invention.

[0094]FIG. 71A is a graph of fluorescence emission versus time for afluorophore being excited at different light intensities, in accordancewith aspects of the invention.

[0095]FIG. 71B is a schematic diagram of an embodiment of a method forincreasing the signal-to-noise ratio of a detected signal by modulationof an exciting light source and demodulation of the detected signal,based on the modulation, in accordance with aspects of the invention.

[0096]FIG. 71C is a pair of graphs of time-dependent measured noise andmeasured signal plus noise without (top) and with (bottom)implementation of the modulation-demodulation method of FIG. 71B in amicrofluidic system, in accordance with aspects of the invention.

[0097]FIG. 71D is a graph of measured fluorescence intensity versus timeprior to and during cycles of exposure of a biotinylated bead to astreptavidin-dye conjugate in a microfluidic system, in accordance withaspects of the invention.

[0098]FIG. 71E is a graph of measured fluorescence intensity versus timeprior to and during exposure of ionomcyin to a retained cell that waspreloaded with a calcium-sensor dye, using the method of FIG. 71B in amicrofluidic system, in accordance with aspects of the invention.

[0099]FIG. 71F is a graph of measured fluorescence intensity versus timeat a position in a microfluidic system prior to and during exposure to adye, in accordance with aspects of the invention.

[0100]FIG. 72 is a time-lapse set of photographic images recordingsize-selective flow of blood cells through a microfluidic system, inaccordance with aspects of the invention.

[0101]FIG. 73 is diagram showing the structure of biotin and its mode ofbinding to streptavidin.

[0102]FIG. 74 is a time-lapse set of photographic images recordinginteraction of specific binding pairs on beads in a microfluidic system,in accordance with aspects of the invention.

[0103]FIG. 75 is a time-lapse set of photographic images recordingstimulation of ion flux in a microfluidic system, in accordance withaspects of the invention.

[0104]FIG. 76 is a time-lapse set of photographic images recordingapoptosis and necrosis in a microfluidic system, in accordance withaspects of the invention.

[0105]FIGS. 77 and 78 are diagrams showing the structures andexcitation/emission spectra for membrane dyes used in the analysis ofExample 22.

[0106]FIG. 79 is a photographic image recording successful staining of acell's membrane in a non-microfluidic environment.

[0107]FIG. 80 is a time-lapse set of photographic images recordingretention of a single cell at a preselected site in a microfluidicsystem, in accordance with aspects of the invention.

[0108]FIG. 81 is a time-lapse set of photographic images recordingretention of a group of cells at a preselected site in a microfluidicsystem, in accordance with aspects of the invention.

[0109]FIG. 82 is a time-lapse set of photographic images recording entryof a fluorescent cell into a retention chamber already holding severalcells, in accordance with aspects of the invention.

[0110]FIG. 83 is a time-lapse set of photographic images recordingfixation and staining of a retained cell in a microfluidic system, inaccordance with aspects of the invention.

[0111]FIG. 84 is a top plan view of a microfluidic system for analyzinga size-selected set of cells, in which the system includes seriallydisposed filtration and retention mechanisms, a perfusion mechanism, anda flow-based detection mechanism, in accordance with aspects of theinvention.

[0112]FIG. 85 is another top plan view of the microfluidic system ofFIG. 84, showing identifying labels for reservoirs and valves, inaccordance with aspects of the invention.

[0113]FIG. 86 is a top plan view of selected portions of the system ofFIG. 84, illustrating selected aspects including a filtration mechanism,in accordance with aspects of the invention.

[0114]FIG. 87 is another top plan view of selected portions of thesystem of FIG. 84, in accordance with aspects of the invention.

[0115]FIG. 88 is yet another top plan view of selected portions of thesystem of FIG. 84, in accordance with aspects of the invention.

[0116]FIG. 89 is a top plan view of a perfusion device for exposingparticles to an array of different reagents or different reagentconcentrations.

[0117]FIGS. 90 through 94 depict a top plan view of a device being usedto measure chemotactic response of cells to a chemoattractant.

[0118]FIG. 95 is a close-up top plan view of a perfusion chamber withassociated valving system.

DETAILED DESCRIPTION

[0119] The invention provides systems, including apparatus, methods, andkits, for the microfluidic manipulation and/or analysis of particles,such as cells, viruses, organelles, beads, and/or vesicles. Theinvention also provides microfluidic mechanisms for carrying out thesemanipulations and analyses. These mechanisms may enable controlledinput, movement/positioning, retention/localization, treatment,measurement, release, and/or output of particles. Furthermore, thesemechanisms may be combined in any suitable order and/or employed for anysuitable number of times within a system. Accordingly, thesecombinations may allow particles to be sorted, cultured, mixed, treated,and/or assayed, among others, as single particles, mixed groups ofparticles, arrays of particles, heterogeneous particle sets, and/orhomogeneous particle sets, among others, in series and/or in parallel.In addition, these combinations may enable microfluidic systems to bereused. Furthermore, these combinations may allow the response ofparticles to treatment to be measured on a shorter time scale than waspreviously possible. Therefore, systems of the invention may allow abroad range of cell and particle assays, such as drug screens, cellcharacterizations, research studies, and/or clinical analyses, amongothers, to be scaled down to microfluidic size. Such scaled-down assaysmay use less sample and reagent, may be less labor intensive, and/or maybe more informative than comparable macrofluidic assays.

[0120] Further aspects of the invention are described in the followingsections: (I) microfluidic systems, (II) physical structures of fluidnetworks, (III) particles, (IV) input mechanisms, (V) positioningmechanisms, (VI) retention mechanisms, (VII) treatment mechanisms,(VIII) measurement mechanisms, (IX) release mechanisms, (X) outputmechanisms, (XI) cell culture mechanisms, (XII) particle-basedmanipulations, and (XIII) examples.

[0121] Microfluidic Systems

[0122] Definitions and Overview

[0123] Particle manipulations and analyses are performed in microfluidicsystems. A microfluidic system generally comprises any system in whichvery small volumes of fluid are stored and manipulated, generally lessthan about 500 μL, typically less than about 100 μL, and more typicallyless than about 10 μL. Microfluidic systems carry fluid in predefinedpaths through one or more microfluidic passages. A microfluidic passagemay have a minimum dimension, generally height or width, of less thanabout 200, 100, or 50 μm. Passages are described in more detail below inSection II.

[0124] Microfluidic systems may include one or more sets of passagesthat interconnect to form a generally closed microfluidic network. Sucha microfluidic network may include one, two, or more openings at networktermini, or intermediate to the network, that interface with theexternal world. Such openings may receive, store, and/or dispense fluid.Dispensing fluid may be directly into the microfluidic network or tosites external the microfluidic system. Such openings generally functionin input and/or output mechanisms, described in more detail in SectionsIV and X below, and may include reservoirs, described in more detail inSection II below.

[0125] Microfluidic systems also may include any other suitable featuresor mechanisms that contribute to fluid, reagent, and/or particlemanipulation or analysis. For example, microfluidic systems may includeregulatory or control mechanisms that determine aspects of fluid flowrate and/or path. Valves and/or pumps that may participate in suchregulatory mechanisms are described in more detail below in Section II.Alternatively, or in addition, microfluidic systems may includemechanisms that determine, regulate, and/or sense fluid temperature,fluid pressure, fluid flow rate, exposure to light, exposure to electricfields, magnetic field strength, and/or the like. Accordingly,microfluidic systems may include heaters, coolers, electrodes, lenses,gratings, light sources, pressure sensors, pressure transducers,microprocessors, microelectronics, and/or so on. Furthermore, eachmicrofluidic system may include one or more features that act as a codeto identify a given system. The features may include any detectableshape or symbol, or set of shapes or symbols, such as black-and-white orcolored barcode, a word, a number, and/or the like, that has adistinctive position, identity, and/or other property (such as opticalproperty).

[0126] Materials

[0127] Microfluidic systems may be formed of any suitable material orcombination of suitable materials. Suitable materials may includeelastomers, such as polydimethylsiloxane (PDMS); plastics, such aspolystyrene, polypropylene, polycarbonate, etc.; glass; ceramics;sol-gels; silicon and/or other metalloids; metals or metal oxides;biological polymers, mixtures, and/or particles, such as proteins(gelatin, polylysine, serum albumin, collagen, etc.), nucleic acids,microorganisms, etc.; and/or the like.

[0128] Exemplary materials for microfluidic systems are described inmore detail in the patent applications listed above underCross-References, which are incorporated herein by reference.

[0129] Methods of Fabrication

[0130] Microfluidic systems, also referred to as chips, may have anysuitable structure. Such systems may be fabricated as a unitarystructure from a single component, or as a multi-component structure oftwo or more components. The two or more components may have any suitablerelative spatial relationship and may be attached to one another by anysuitable bonding mechanism.

[0131] In some embodiments, two or more of the components may befabricated as relatively thin layers, which may be disposedface-to-face. The relatively thin layers may have distinct thickness,based on function. For example, the thickness of some layers may beabout 10 to 250 μm, 20 to 200 μm, or about 50 to 150 μm, among others.Other layers may be substantially thicker, in some cases providingmechanical strength to the system. The thicknesses of such other layersmay be about 0.25 to 2 cm, 0.4 to 1.5 cm, or 0.5 to 1 cm, among others.One or more additional layers may be a substantially planar layer thatfunctions as a substrate layer, in some cases contributing a floorportion to some or all microfluidic passages.

[0132] Components of a microfluidic system may be fabricated by anysuitable mechanism, based on the desired application for the system andon materials used in fabrication. For example, one or more componentsmay be molded, stamped, and/or embossed using a suitable mold. Such amold may be formed of any suitable material by micromachining, etching,soft lithography, material deposition, cutting, and/or punching, amongothers. Alternatively, or in addition, components of a microfluidicsystem may be fabricated without a mold by etching, micromachining,cutting, punching, and/or material deposition.

[0133] Microfluidic components may be fabricated separately, joined, andfurther modified as appropriate. For example, when fabricated asdistinct layers, microfluidic components may be bonded, generallyface-to-face. These separate components may be surface-treated, forexample, with reactive chemicals to modify surface chemistry, withparticle binding agents, with reagents to facilitate analysis, and/or soon. Such surface-treatment may be localized to discrete portions of thesurface or may be relatively nonlocalized. In some embodiments, separatelayers may be fabricated and then punched and/or cut to produceadditional structure. Such punching and/or cutting may be performedbefore and/or after distinct components have been joined.

[0134] Exemplary methods for fabricating microfluidic systems aredescribed in more detail in the patent applications identified aboveunder Cross-References, which are incorporated herein by reference.

[0135] Physical Structures of Fluid Networks

[0136] Overview

[0137] Microfluidic systems may include any suitable structure(s) forthe integrated manipulation of small volumes of fluid, including movingand/or storing fluid, and particles associated therewith, for use inparticle assays. The structures may include passages, reservoirs, and/orregulators, among others.

[0138] Passages

[0139] Passages generally comprise any suitable path, channel, or ductthrough, over, or along which materials (e.g., fluid, particles, and/orreagents) may pass in a microfluidic system. Collectively, a set offluidically communicating passages, generally in the form of channels,may be referred to as a microfluidic network. In some cases, passagesmay be described as having surfaces that form a floor, a roof, andwalls. Passages may have any suitable dimensions and geometry, includingwidth, height, length, and/or cross-sectional profile, among others, andmay follow any suitable path, including linear, circular, and/orcurvilinear, among others. Passages also may have any suitable surfacecontours, including recesses, protrusions, and/or apertures, and mayhave any suitable surface chemistry or permeability at any appropriateposition within a channel. Suitable surface chemistry may includesurface modification, by addition and/or treatment with a chemicaland/or reagent, before, during, and/or after passage formation.

[0140] In some cases, passages, and particularly channels, may bedescribed according to function. For example, passages may be describedaccording to direction of material flow in a particular application,relationship to a particular reference structure, and/or type ofmaterial carried. Accordingly, passages may be inlet passages (orchannels), which generally carry materials to a site, and outletpassages (or channels), which generally carry materials from a site. Inaddition, passages may be referred to as particle passages (orchannels), reagent passages (or channels), focusing passages (orchannels), perfusion passages (or channels), waste passages (orchannels), and/or the like.

[0141] Passages may branch, join, and/or dead-end to form any suitablemicrofluidic network. Accordingly, passages may function in particlepositioning, sorting, retention, treatment, detection, propagation,storage, mixing, and/or release, among others.

[0142] Further aspects of passages are included throughout this DetailedDescription, and in the patent applications identified above underCross-References, which are incorporated herein by reference.

[0143] Reservoirs

[0144] Reservoirs generally comprise any suitable receptacle or chamberfor storing materials (e.g., fluid, particles and/or reagents), before,during, between, and/or after processing operations (e.g., measurementand/or treatment). Reservoirs, also referred to as wells, may includeinput, intermediate, and/or output reservoirs. Input reservoirs maystore materials (e.g., fluid, particles, and/or reagents) prior toinputting the materials to a microfluidic network(s) portion of a chip.By contrast, intermediate reservoirs may store materials during and/orbetween processing operations. Finally, output reservoirs may storematerials prior to outputting from the chip, for example, to an externalprocessor or waste, or prior to disposal of the chip.

[0145] Further aspects of reservoirs are included in the patentapplications identified above under Cross-References, which areincorporated herein by reference.

[0146] Regulators

[0147] Regulators generally comprise any suitable mechanism forgenerating and/or regulating movement of materials (e.g., fluid,particles, and/or reagents). Suitable regulators may include valves,pumps, and/or electrodes, among others. Regulators may operate byactively promoting flow and/or by restricting active or passive flow.Suitable functions mediated by regulators may include mixing, sorting,connection (or isolation) of fluidic networks, and/or the like.

[0148] Further aspects of regulators, particularly the structure,fabrication, and operation of valves and pumps, are included in thepatent applications identified above under Cross-References, which areincorporated herein by reference, and in Section XIII, particularlyExample 8.

[0149] Particles

[0150] Overview

[0151] Microfluidic systems may be used to manipulate and/or analyzeparticles. A particle generally comprises any object that is smallenough to be inputted and manipulated within a microfluidic network inassociation with fluid, but that is large enough to be distinguishablefrom the fluid. Particles, as used here, typically are microscopic ornear-microscopic, and may have diameters of about 0.005 to 100 μm, 0.1to 50 μm, or about 0.5 to 30 μm. Alternatively, or in addition,particles may have masses of about 10⁻²⁰ to 10⁻⁵ grams, 10⁻¹⁶ to 10⁻⁷grams, or 10 ⁻¹⁴ to 10⁻⁸ grams. Exemplary particles may include cells,viruses, organelles, beads, and/or vesicles, and aggregates thereof,such as dimers, trimers, etc.

[0152] Cells

[0153] Overview

[0154] Cells, as used here, generally comprise any self-replicating,membrane-bounded biological entity, or any nonreplicating,membrane-bounded descendant thereof. Nonreplicating descendants may besenescent cells, terminally differentiated cells, cell chimeras,serum-starved cells, infected cells, nonreplicating mutants, anucleatecells, etc.

[0155] Cells used as particles in microfluidic systems may have anysuitable origin, genetic background, state of health, state of fixation,membrane permeability, pretreatment, and/or population purity, amongothers. Origin of cells may be eukaryotic, prokaryotic, archae, etc.,and may be from animals, plants, fungi, protists, bacteria, and/or thelike. Cells may be wild-type; natural, chemical, or viral mutants;engineered mutants (such as transgenics); and/or the like. In addition,cells may be growing, quiescent, senescent, transformed, and/orimmortalized, among others, and cells may be fixed and/or unfixed.Living or dead, fixed or unfixed cells may have intact membranes, and/orpermeabilized/disrupted membranes to allow uptake of ions, labels, dyes,ligands, etc., or to allow release of cell contents. Cells may have beenpretreated before introduction into a microfluidic system by anysuitable processing steps. Such processing steps may include modulatortreatment, transfection (including infection, injection, particlebombardment, lipofection, coprecipitate transfection, etc.), processingwith assay reagents, such as dyes or labels, and/or so on. Furthermore,cells may be a monoculture, generally derived as a clonal populationfrom a single cell or a small set of very similar cells; may bepresorted by any suitable mechanism such as affinity binding, FACS, drugselection, etc.; and/or may be a mixed or heterogeneous population ofdistinct cell types.

[0156] Eukaryotic Cells

[0157] Eukaryotic cells, that is, cells having one or more nuclei, oranucleate derivatives thereof, may be obtained from any suitable source,including primary cells, established cells, and/or patient samples. Suchcells may be from any cell type or mixture of cell types, from anydevelopmental stage, and/or from any genetic background. Furthermore,eukaryotic cells may be adherent and/or nonadherent. Such cells may befrom any suitable eukaryotic organism including animals, plants, fungi,and/or protists.

[0158] Eukaryotics cells may be from animals, that is, vertebrates orinvertebrates. Vertebrates may include mammals, that is, primates (suchas humans, apes, monkeys, etc.) or nonprimates (such as cows, horses,sheep, pigs, dogs, cats, marsupials, rodents, and/or the like).Nonmammalian vertebrates may include birds, reptiles, fish, (such astrout, salmon, goldfish, zebrafish, etc.), and/or amphibians (such asfrogs of the species Xenopus, Rana, etc.). Invertebrates may includearthropods (such as arachnids, insects (e.g., Drosophila), etc.),mollusks (such as clams, snails, etc.), annelids (such as earthworms,etc.), echinoderms (such as various starfish, among others),coelenterates (such as jellyfish, coral, etc.), porifera (sponges),platyhelminths (tapeworms), nemathelminths (flatworms), etc.

[0159] Eukaryotic cells may be from any suitable plant, such asmonocotyledons, dicotyledons, gymnosperms, angiosperms, ferns, mosses,lichens, and/or algae, among others. Exemplary plants may include plantcrops (such as rice, corn, wheat, rye, barley, potatoes, etc.), plantsused in research (e.g., Arabadopsis, loblolly pine, etc.), plants ofhorticultural values (ornamental palms, roses, etc.), and/or the like.

[0160] Eukaryotic cells may be from any suitable fungi, includingmembers of the phyla Chytridiomycota, Zygomycota, Ascomycota,Basidiomycota, Deuteromycetes, and/or yeasts. Exemplary fungi mayinclude Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoralis, Neurospora crassa, mushrooms, puffballs, imperfect fungi,molds, and/or the like.

[0161] Eukaryotic cells may be from any suitable protists (protozoans),including amoebae, ciliates, flagellates, coccidia, microsporidia,and/or the like. Exemplary protists may include Giardia lamblia,Entamoeba. histolytica, Cryptosporidium, and/or N. fowleri, amongothers.

[0162] Particles may include eukaryotic cells that are primary, that is,taken directly from an organism or nature, without subsequent extendedculture in vitro. For example, the cells may be obtained from a patientsample, such as whole blood, packed cells, white blood cells, urine,sputum, feces, mucus, spinal fluid, tumors, diseased tissue, bonemarrow, lymph, semen, pleural fluid, a prenatal sample, an aspirate, abiopsy, disaggregated tissue, epidermal cells, keratinocytes,endothelial cells, smooth muscle cells, skeletal muscle cells, neuralcells, renal cells, prostate cells, liver cells, stem cells,osteoblasts, and/or the like. Similar samples may be manipulated andanalyzed from human volunteers, selected members of the humanpopulation, forensic samples, animals, plants, and/or natural sources(water, soil, air, etc.), among others.

[0163] Alternatively, or in addition, particles may include establishedeukaryotic cells. Such cells may be immortalized and/or transformed byany suitable treatment, including viral infection, nucleic acidtransfection, chemical treatment, extended passage and selection,radiation exposure, and/or the like. Such established cells may includevarious lineages such as neuroblasts, neurons, fibroblasts, myoblasts,myotubes, chondroblasts, chondrocytes, osteoblasts, osteocytes,cardiocytes, smooth muscle cells, epithelial cells, keratinocytes,kidney cells, liver cells, lymphocytes, granulocytes, and/ormacrophages, among others. Exemplary established cell lines may includeRat-1, NIH 3T3, HEK 293, COS1, COS7, CV-1, C2C12, MDCK, PC12, SAOS,HeLa, Schneider cells, Junkat cells, SL2, and/or the like.

[0164] Prokaryotic Cells

[0165] Particles may be prokaryotic cells, that is, self-replicating,membrane-bounded microorganisms that lack membrane-bound organelles, ornonreplicating descendants thereof. Prokaryotic cells may be from anyphyla, including Aquificae, Bacteroids, Chlorobia, Chrysogenetes,Cyanobacteria, Fibrobacter, Firmicutes, Flavobacteria, Fusobacteria,Proteobacteria, Sphingobacteria, Spirochaetes, Thermomicrobia, and/orXenobacteria, among others. Such bacteria may be gram-negative,gram-positive, harmful, beneficial, and/or pathogenic. Exemplaryprokaryotic cells may include E. coli, S. typhimurium, B. subtilis, S.aureus, C. perfringens, V. parahaemolyticus, and/or B. anthracis, amongothers.

[0166] Viruses

[0167] Viruses may be manipulated and/or analyzed as particles inmicrofluidic systems. Viruses generally comprise anymicroscopic/submicroscopic parasites of cells (animals, plants, fungi,protists, and/or bacteria) that include a protein and/or membrane coatand that are unable to replicate without a host cell. Viruses mayinclude DNA viruses, RNA viruses, retroviruses, virions, viroids,prions, etc. Exemplary viruses may include HIV, RSV, rabies, hepatitisvirus, Epstein-Barr virus, rhinoviruses, bacteriophages, prions thatcause various diseases (CJD (Creutzfeld-Jacob disease, kuru, GSS(Gerstmann-Straussler-Scheinker syndrome), FFI (Fatal FamilialInsomnia), Alpers syndrome, etc.), and/or the like.

[0168] Organelles

[0169] Organelles may be manipulated and/or analyzed in microfluidicsystems. Organelles generally comprise any particulate component of acell. For example, organelles may include nuclei, Golgi apparatus,lysosomes, endosomes, mitochondria, peroxisomes, endoplasmic reticulum,phagosomes, vacuoles, chloroplasts, etc.

[0170] Beads

[0171] Particle assays may be performed with beads. Beads generallycomprise any suitable manufactured particles. Beads may be manufacturedfrom inorganic materials, or materials that are synthesized chemically,enzymatically and/or biologically. Furthermore, beads may have anysuitable porosity and may be formed as a solid or as a gel. Suitablebead compositions may include plastics (e.g., polystyrene), dextrans,glass, ceramics, sol-gels, elastomers, silicon, metals, and/orbiopolymers (proteins, nucleic acids, etc.). Beads may have any suitableparticle diameter or range of diameters. Accordingly, beads may be asubstantially uniform population with a narrow range of diameters, orbeads may be a heterogeneous population with a broad range of diameters,or two or more distinct diameters.

[0172] Beads maybe associated with any suitable materials. The materialsmay include compounds, polymers, complexes, mixtures, phages, viruses,and/or cells, among others. For example, the beads may be associatedwith a member of a specific binding pair (see Section VI), such as areceptor, a ligand, a nucleic acid, a member of a compound library,and/or so on. Beads may be a mixture of distinct beads, in some casescarrying distinct materials. The distinct beads may differ in anysuitable aspect(s), such as size, shape, an associated code, and/ormaterial carried by the beads. In some embodiments, the aspect mayidentify the associated material. Codes are described further in SectionXII below.

[0173] Vesicles

[0174] Particles may be vesicles. Vesicles generally comprise anynoncellularly derived particle that is defined by a lipid envelope.Vesicles may include any suitable components in their envelope orinterior portions. Suitable components may include compounds, polymers,complexes, mixtures, aggregates, and/or particles, among others.Exemplary components may include proteins, peptides, small compounds,drug candidates, receptors, nucleic acids, ligands, and/or the like.

[0175] Input Mechanisms

[0176] Overview

[0177] Microfluidic systems may include one or more input mechanismsthat interface with the microfluidic network(s). An input mechanismgenerally comprises any suitable mechanism for inputting material(s)(e.g., particles, fluid, and/or reagents) to a microfluidic network of amicrofluidic chip, including selective (that is, component-by-component)and/or bulk mechanisms.

[0178] Internal/External Sources

[0179] The input mechanism may receive material from internal sources,that is, reservoirs that are included in a microfluidic chip, and/orexternal sources, that is, reservoirs that are separate from, orexternal to, the chip.

[0180] Input mechanisms that input materials from internal sources mayuse any suitable receptacle to store and dispense the materials.Suitable receptacles may include a void formed in the chip. Such voidsmay be directly accessible from outside the chip, for example, through ahole extending from fluidic communication with a fluid network to anexternal surface of the chip, such as the top surface. The receptaclesmay have a fluid capacity that is relatively large compared to the fluidcapacity of the fluid network, so that they are not quickly exhausted.For example, the fluid capacity may be at least about 1, 5, 10, 25, 50,or 100 μL. Accordingly, materials may be dispensed into the receptaclesusing standard laboratory equipment, if desired, such as micropipettes,syringes, and the like.

[0181] Input mechanisms that input materials from external sources alsomay use any suitable receptacle and mechanism to store and dispense thematerials. However, if the external sources input materials directlyinto the fluid network, the external sources may need to interfaceeffectively with the fluid network, for example, using contact and/ornoncontact dispensing mechanisms. Accordingly, input mechanisms fromexternal sources may use capillaries or needles to direct fluidprecisely into the fluid network. Alternatively, or in addition, inputmechanisms from external sources may use a noncontact dispensingmechanism, such as “spitting,” which may be comparable to the action ofan inkjet printer. Furthermore, input mechanisms from external sourcesmay use ballistic propulsion of particles, for example, as mediated by agene gun.

[0182] Facilitating Mechanisms

[0183] The inputting of materials into the microfluidics system may befacilitated and/or regulated using any suitable facilitating mechanism.Such facilitating mechanisms may include gravity flow, for example, whenan input reservoir has greater height of fluid than an output reservoir.Facilitating mechanisms also may include positive pressure to pushmaterials into the fluidic network, such as mechanical or gas pressure,or centrifugal force; negative pressure at an output mechanism to drawfluid toward the output mechanism; and/or a positioning mechanism actingwithin the fluid network. The positioning mechanism may include a pumpand/or an electrokinetic mechanism. Positioning mechanisms are furtherdescribed below, in Section V. In some embodiments, the facilitatingmechanism may include a suspension mechanism to maintain particles suchas cells in suspension prior to inputting, for example, as described inExample 7.

[0184] Positioning Mechanisms

[0185] Overview

[0186] Microfluidic systems may include one or more positioningmechanisms. A positioning mechanism generally comprises any mechanismfor placing particles at preselected positions on the chip afterinputting, for example, for retention, growth, treatment, and/ormeasurement, among others. Positioning mechanisms may be categorizedwithout limitation in various ways, for example, to reflect theirorigins and/or operational principles, including direct and/or indirect,fluid-mediated and/or non-fluid-mediated, external and/or internal, andso on. These categories are not mutually exclusive. Thus, a givenpositioning mechanism may position a particle in two or more ways; forexample, electric fields may position a particle directly (e.g., viaelectrophoresis) and indirectly (e.g., via electroosmosis).

[0187] The positioning mechanisms may act to define particle positionlongitudinally and/or transversely. The term “longitudinal position”denotes position parallel to or along the long axis of a microfluidicchannel and/or a fluid flow stream within the channel. In contrast, theterm “transverse position” denotes position orthogonal to the long axisof a channel and/or an associated main fluid flow stream. Bothlongitudinal and transverse positions may be defined locally, byequating “long axis” with “tangent” in curved channels.

[0188] The positioning mechanisms may be used alone and/or incombination. If used in combination, the mechanisms may be used serially(i.e., sequentially) and/or in parallel (i.e., simultaneously). Forexample, an indirect mechanism such as fluid flow may be used for roughpositioning, and a direct mechanism such as optical tweezers may be usedfor final positioning (and/or subsequent retention, as describedelsewhere).

[0189] The remainder of this section describes without limitation avariety of exemplary positioning mechanisms, sorted roughly as directand indirect mechanisms.

[0190] Direct Positioning Mechanisms

[0191] Direct positioning mechanisms generally comprise any mechanismsin which a force acts directly on a particle(s) to position theparticle(s) within a microfluidic network. Direct positioning mechanismsmay be based on any suitable mechanism, including optical, electrical,magnetic, and/or gravity-based forces, among others. Optical positioningmechanisms use light to mediate or at least facilitate positioning ofparticles. Suitable optical positioning mechanisms include “opticaltweezers,” which use an appropriately focused and movable light sourceto impart a positioning force on particles. Electrical positioningmechanisms use electricity to position particles. Suitable electricalmechanisms include “electrokinesis,” that is, the application of voltageand/or current across some or all of a microfluidic network, which may,as mentioned above, move charged particles directly (e.g., viaelectrophoresis) and/or indirectly, through movement of ions in fluid(e.g., via electroosmosis). Magnetic positioning mechanisms usemagnetism to position particles based on magnetic interactions. Suitablemagnetic mechanisms involve applying a magnetic field in or around afluid network, to position particles via their association withferromagnetic and/or paramagnetic materials in, on, or about theparticles. Gravity-based positioning mechanisms use the force of gravityto position particles, for example, to contact adherent cells with asubstrate at positions of cell culture.

[0192] Indirect Positioning Mechanisms

[0193] Indirect positioning mechanisms generally comprise any mechanismsin which a force acts indirectly on a particle(s), for example, viafluid, to move the particle(s) within a microfluidic network,longitudinally and/or transversely.

[0194] Longitudinal Indirect Positioning Mechanisms

[0195] Longitudinal indirect positioning mechanisms generally may becreated and/or regulated by fluid flow along channels and/or otherpassages. Accordingly, longitudinal positioning mechanisms may befacilitated and/or regulated by valves and/or pumps that regulate flowrate and/or path. In some cases, longitudinal positioning mechanisms maybe facilitated and/or regulated by electroosmotic positioningmechanisms. Alternatively, or in addition, longitudinal positioningmechanisms may be input-based, that is, facilitated and/or regulated byinput mechanisms, such as pressure or gravity-based mechanisms,including a pressure head created by unequal heights of fluid columns.

[0196] Transverse Indirect Positioning Mechanisms

[0197] Transverse indirect positioning mechanisms generally may becreated and/or regulated by fluid flow streams at channel junctions,laterally disposed regions of reduced fluid flow, and/or channel bends.Channel junctions may be unifying sites or dividing sites, based on thenumber of channels that carry fluid to the sites relative to the numberthat carry fluid away from the sites. Transverse indirect positioningmechanisms may be based on laminar flow, stochastic partitioning, and/orcentrifugal force, among others.

[0198] Laminar Flow-Based Transverse Positioning Mechanisms

[0199] Transverse positioning of particles and/or reagents in amicrofluidic system may be mediated at least in part by a laminarflow-based mechanism. Laminar flow-based mechanisms generally compriseany positioning mechanism in which the position of an input flow streamwithin a channel is determined by the presence, absence, and/or relativeposition(s) of additional flow streams within the channel. Such laminarflow-based mechanisms may be defined by a channel junction(s) that is aunifying site, at which inlet flow streams from two, three, or morechannels, flowing toward the junction, unify to form a smaller number ofoutlet flow streams, preferably one, flowing away from the junction. Dueto the laminar flow properties of flow streams on a microfluidic scale,the unifying site may maintain the relative distribution of inlet flowstreams after they unify as laminar outlet flow streams. Accordingly,particles and/or reagents may remain localized to any selected one ormore of the laminar flow streams, based on which inlet channels carryparticles and/or reagents, thus positioning the particles and/orreagents transversely.

[0200] The relative size (or flow rate) and position of each inlet flowstream may determine both transverse position and relative width of flowstreams that carry particles and/or reagents. For example, an inlet flowstream for particles/reagents that is relatively small (narrow), flankedby two larger (wider) flow streams, may occupy a narrow central positionin a single outlet channel. By contrast, an inlet flow stream forparticles/reagents that is relatively large (wide), flanked by acomparably sized flow stream and a smaller (narrower) flow stream, mayoccupy a wider position that is biased transversely toward the smallerflow stream. In either case, the laminar flow-based mechanism may becalled a focusing mechanism, because the particles/reagents are“focused” to a subset of the cross-sectional area of outlet channels.Laminar flow-based mechanisms may be used to individually addressparticles and/or reagents to plural distinct retention sites. Exemplarylaminar flow-based positioning mechanisms are further described below,in Examples 2-4, 7, 9, 11, and 26, among others.

[0201] A laminar flow-based mechanism may be a variable mechanism tovary the transverse position of particles/reagents. As described above,the relative contribution of each inlet flow stream may determine thetransverse position of particles/reagents flow streams. Altered flow ofany inlet flow stream may vary its contribution to the outlet flowstream(s), shifting particles/reagents flow streams accordingly. In anextreme case, referred to as a perfusion mechanism, a reagent (orparticle) flow stream may be moved transversely, either in contact with,or spaced from, retained particles (reagents), based on presence orabsence of flow from an adjacent inlet flow stream. Such a mechanismalso may be used to effect variable or regulated transverse positioningof particles, for example, to direct particles to retention sites havingdifferent transverse positions. Exemplary variable or regulatedtransverse positioning mechanisms, referred to as perfusion mechanisms,are further described below, in Examples 2-4, 6, 7, 11, and 26, amongothers.

[0202] Stochastic Transverse Positioning Mechanisms

[0203] Transverse positioning of particles and/or reagents in amicrofluidic system may be mediated at least in part by a stochastic (orportioned flow) positioning mechanism. Stochastic transverse positioningmechanisms generally comprise any positioning mechanism in which an atleast partially randomly selected subset of inputted particles orreagent is distributed laterally away from a main flow stream to aregion of reduced fluid flow within a channel (or, potentially, to adistinct channel). The region of reduced flow may promote particleretention, treatment, detection, minimize particle damage, and/orpromote particle contact with a substrate. Stochastic positioningmechanisms may be determined by dividing flow sites and/or locallywidened channels, among others.

[0204] Dividing flow sites may effect stochastic positioning by formingregions of reduced fluid flow rate. Dividing flow sites generallyinclude any channel junction at which inlet flow streams from one(preferably) or more inlet channels are divided into a greater number ofoutlet channels, including two, three, or more, channels. Such dividingsites may deliver a subset of particles, which may be selectedstochastically and/or based on a property of the particles (such asmass), to a region of reduced flow rate or quasi-stagnant flow formed ator near the junction. The fraction of particles represented by thesubset may be dependent upon the relative flow directions of the outletchannels relative to the inlet channels. These flow directions may begenerally orthogonal to an inlet flow stream, being directed in oppositedirections, to form a “T-junction.” Alternatively, outlet flowdirections may form angles of less than and/or greater than 90°.Exemplary reduced-velocity, dividing-flow positioning mechanisms arefurther described below, in Examples 1, 2, 3, 4, 6, 7, and 26, amongothers.

[0205] The dividing-flow positioning mechanism, with two or more outletchannels, may be used as a portioned-flow mechanism. Specifically,fluid, particles, and/or reagents carried to the channel junction may beportioned according to fluid flow through the two or more outletchannels. Accordingly, the fractional number or volume of particles orreagent that enters the two or more channels may be regulated by therelative sizes of the channels and/or the flow rate of fluid through thechannels, which in turn may be regulated by valves, or other suitableflow regulatory-mechanisms. In a first set of embodiments, outletchannels may be of very unequal sizes, so that only a small fraction ofparticle and/or reagents are directed to the smaller channel. In asecond set of embodiments, valves may be used to forms desired dilutionsof reagents. In a third set of embodiments, valves may be used toselectively direct particles to one of two or more fluid paths. Examplesof these three sets of embodiments are further described below, inExamples 11, 8, and 7, respectively.

[0206] Locally widened channels may promote stochastic positioning byproducing regions of decreased flow rate lateral to a main flow stream.The decreased flow rate may deposit a subset of inputted particles at aregion of decreased flow rate. Such widened channels may includenonlinear channels that curve or bend at an angle. Alternatively, or inaddition, widened regions may be formed by recesses formed in a channelwall(s), chambers that intersect channels, and/or the like, particularlyat the outer edge of a curved or bent channel. Exemplary locally widenedchannels that promote stochastic transverse positioning are describedfurther in Example 10.

[0207] Centrifugal-force-based Transverse Positioning Mechanisms

[0208] Transverse positioning of particles and/or reagents also may bemediated at least in part by a centrifugal positioning mechanism. Incentrifugal positioning mechanisms, particles may experience acentrifugal force determined by a change in velocity, for example, bymoving through a bend in a fluid path. Size and/or density of particlesmay determine the rate of velocity change, distributing distinct sizesand/or densities of particle to distinct transverse positions. Exemplarycentrifugal positioning mechanisms are further described below, inExample 9.

[0209] Retention Mechanisms

[0210] Overview

[0211] Microfluidic systems may include one or more retentionmechanisms. A retention mechanism generally comprises any suitablemechanism for retaining (or holding, capturing, or trapping) particlesat preselected positions or regions of microfluidic networks, includingsingle or plural mechanisms, operating in series and/or in parallel.Retention mechanisms may act to overcome the positioning force exertedby fluid flow. Furthermore, retention mechanisms, also referred to ascapture or trapping mechanisms, may retain any suitable number ofparticles, including single particles or groups/populations ofparticles. Suitable retention mechanisms may be based on physicalbarriers coupled with flow, chemical interactions, vacuum forces, fluidflow in a loop, gravity, centrifugal forces, magnetic forces, electricalforces, and/or optically generated forces, among others.

[0212] Retention mechanisms may be selective or nonselective. Selectivemechanisms may be fractionally selective, that is, retaining less thanall (a subset of) inputted particles. Fractionally selective mechanismsmay rely at least in part on stochastic positioning mechanisms, such asthat exemplified in Example 2. Alternatively, or in addition, selectivemechanisms may be particle-dependent, that is, retaining particles basedon one or more properties of the inputted particle, such as size,surface chemistry, density, magnetic character, electrical charge,optical property (such as refractive index), and/or the like.

[0213] Physical Barrier-Based Retention Mechanisms

[0214] Retention mechanisms may be based at least partially on particlecontact with any suitable physical barrier(s) disposed in a microfluidicnetwork. Such particle-barrier contact generally restricts longitudinalparticle movement along the direction of fluid flow, producingflow-assisted retention. Flow-assisted particle-barrier contact also mayrestrict side-to-side/orthogonal (transverse) movement. Suitablephysical barriers may be formed by protrusions that extend inward fromany portion of a channel or other passage (that is, walls, roof, and/orfloor). For example, the protrusions may be fixed and/or movable,including columns, posts, blocks, bumps, walls, and/orpartially/completely closed valves, among others. Some physicalbarriers, such as valves, may be movable or regulatable. Alternatively,or in addition, a physical barrier may be defined by a recess(es) formedin a channel or other passage, or by a fluid-permeable membrane. Otherphysical barriers may be formed based on the cross-sectional dimensionsof passages. For example, size-selective channels may retain particlesthat are too large to enter the channels. (Size-selective channels alsomay be referred to as filter channels, microchannels, orparticle-restrictive or particle-selective channels.)

[0215] Further aspects of physical barriers and size-selective channelsare described below in Section XIII, and in the patent applicationslisted in the Cross-References, which are incorporated herein byreference.

[0216] Chemical Retention Mechanisms

[0217] Chemical retention mechanisms may retain particles based onchemical interactions. The chemical interactions may be covalent and/ornoncovalent interactions, including ionic, electrostatic, hydrophobic,van der Waals, and/or metal coordination interactions, among others.Chemical interactions may retain particles selectively and/ornonselectively. Selective and nonselective retention may be based onspecific and/or nonspecific chemical interactions between particles andpassage surfaces.

[0218] Chemical interactions may be specific. Specific mechanisms mayuse specific binding pairs (SBPs), for example, with first and secondSBP members disposed on particles and passage surfaces, respectively.Exemplary SBPs may include biotin/avidin, antibody/antigen,lectin/carbohydrate, etc. These and additional exemplary SBPs are listedbelow in Table 1, with the designations of first and second beingarbitrary. SBP members may be disposed locally within microfluidicnetworks before, during and/or after formation of the networks. Forexample, surfaces of a substrate and/or a fluid layer component may belocally modified by adhesion/attachment of a SBP member before thesubstrate and fluid layer component are joined. Alternatively, or inaddition, an SBP member may be locally associated with a portion of amicrofluidic network after the network has been formed, for example, bylocal chemical reaction of the SBP member with the network (such ascatalyzed by local illumination with light). TABLE 1 RepresentativeSpecific Binding Pairs First SBP Member Second SBP Member Antigenantibody Biotin avidin or streptavidin Carbohydrate lectin orcarbohydrate receptor DNA antisense DNA or DNA-binding protein enzymesubstrate or enzyme inhibitor Histidine NTA (nitrilotriacetic acid) IgGprotein A or protein G RNA antisense RNA

[0219] Chemical interactions also may be relatively nonspecific.Nonspecific interaction mechanisms may rely on local differences in thesurface chemistry of microfluidic networks. Such local differences maybe created before, during and/or after passage/microfluidic networkformation, as described above. The local differences may result fromlocalized chemical reactions, for example, to create hydrophobic orhydrophilic regions, and/or localized binding of materials. The boundmaterials may include poly-L-lysine, poly-D-lysine, polyethylenimine,albumin, gelatin, collagen, laminin, fibronectin, entactin, vitronectin,fibrillin, elastin, heparin, keratan sulfate, heparan sulfate,chondroitin sulfate, hyaluronic acid, and/or extracellular matrixextracts/mixtures, among others.

[0220] Other Retention Mechanisms

[0221] Other retention mechanisms may be used alternatively, or inaddition to, physical barrier-based and/or chemical interaction-basedretention. Some or all of these mechanisms, and/or the mechanismsdescribed above, may rely at least partially on friction betweenparticles and passages to assist retention.

[0222] Retention mechanisms may be based on vacuum forces, fluid flow,and/or gravity. Vacuum-based retention mechanisms may exert forces thatpull particles into tighter contact with passage surfaces, for example,using a force directed outwardly from a channel. Application of avacuum, and/or particle retention, may be assisted by anaperture/orifice in the wall of a channel or other passage. By contrast,fluid flow-based retention mechanisms may produce fluid flow paths, suchas loops, that retain particles. These fluid flow paths may be formed bya closed channel-circuit having no outlet (e.g., by valve closure andactive pumping), and/or by an eddy, such as that produced by generallycircular fluid-flow within a recess. Gravity-based retention mechanismsmay hold particles against the bottom surfaces of passages, thuscombining with friction to restrict particle movement. Gravity-basedretention may be facilitated by recesses and/or reduced fluid flowrates. Further aspects of vacuum-based and fluid flow-based retentionmechanisms are described below in Examples 11 and 12, and Example 10,respectively.

[0223] Retention mechanisms may be based on centrifugal forces, magneticforces, and/or optically generated forces. Retention mechanisms based oncentrifugal force may retain particles by pushing the particle againstpassage surfaces, typically by exerting a force on the particles that isgenerally orthogonal to fluid flow. Such forces may be exerted bycentrifugation of a microfluidic chip and/or by particle movement withina fluid flow path (see Example 9). Magnetic force-based retentionmechanisms may retain particles using magnetic fields, generatedexternal and/or internal to a microfluidic system. The magnetic fieldmay interact with ferromagnetic and/or paramagnetic portions ofparticles. For example, beads may be formed at least partially offerromagnetic materials, or cells may include surface-bound orinternalized ferromagnetic particles. Electrical force-based retentionmechanisms may retain charged particles and/or populations usingelectrical fields. By contrast, retention mechanisms that operate basedon optically generated forces may use light to retain particles. Suchmechanisms may operate based on the principal of optical tweezers, amongothers.

[0224] Another form of retention mechanism is a blind-fill channel,where a channel has a inlet, but no outlet, either fixedly ortransiently. For example, when the microfluidic device is made from agas permeable material, such as PDMS, gas present in a dead-end channelcan escape, or be forced out of the channel through the gas permeablematerial when urged out by the inflow of liquid through the inlet. Thisis a preferred example of blind-filling. Blind-filling can be used witha channel or chamber that has an inlet, and an outlet that is gated orvalved by a valve. In this example, blind filling of a gas filledchannel or chamber occurs when the outlet valve is closed while fillingthe channel or chamber through the inlet. If the inlet also has a valve,that valve can then be closed after the blind fill is complete, and theoutlet can then be opened to expose the channel or chamber contents toanother channel or chamber. If a third inlet is in communication withthe channel or chamber, that third inlet can introduce another fluid,gas or liquid, into the channel or chamber to expel the blind-filledliquid to be expelled from the channel or chamber in a measured amount.The result is similar to a sample loop system of an HPLC.

[0225] Further aspects of retention mechanisms are described in SectionsV and XIII.

[0226] Treatment Mechanisms

[0227] Overview

[0228] Treatment mechanisms generally comprise any suitable mechanismsfor exposing a particle(s) to a reagent(s) and/or a physicalcondition(s), including fluid-mediated and non-fluid-mediatedmechanisms.

[0229] Reagents

[0230] Particles may be exposed to reagents. A reagent generallycomprises any chemical substance(s), compound(s), ion(s), polymer(s),material(s), complex(es), mixture(s), aggregate(s), and/or biologicalparticle(s), among others, that contacts a particle or particlepopulation in a microfluidic system. Reagents may play a role inparticle analysis, including operating as chemical/biological modulators(interaction reagents), detection/assay reagents, solvents, buffers,media, washing solutions, and/or so on.

[0231] Chemical modulators or biological modulators may include anyreagent that is being tested for interaction with particles. Interactiongenerally includes specific binding to particles and/or any detectablegenotypic and/or phenotypic effect on particles (or modulators). Furtheraspects of interactions and genotypic/phenotypic effects that may besuitable are described below in Section XII.

[0232] Chemical modulators may include ligands that interact withreceptors (e.g., antagonists, agonists, hormones, etc.). Ligands may besmall compounds, peptides, proteins, carbohydrates, lipids, etc. Furtheraspects of ligands and receptors, and their use in measuringinteraction, or effects on signal transduction pathways, are describedbelow in Section XII.

[0233] Alternatively, or in addition, chemical modulators may be nucleicacids. The nucleic acids may be DNA, RNA, peptide nucleic acids,modified nucleic acids, and/or mixtures thereof, and may be single,double, and/or triple-stranded. The nucleic acids may be produced bychemical synthesis, enzymatic synthesis, and/or biosynthesis, and may beplasmids, fragments, sense/antisense expression vectors, reporter genes,vectors for genomic integration/modification (such as targeting nucleicacids/vectors (for knockout/-down/-in)), viral vectors, antisenseoligonucleotides, dsRNA, siRNA, nucleozymes, and/or the like. Nucleicacid reagents may also include transfection reagents to promote uptakeof the nucleic acids by cells, such as lipid reagents (e.g.,lipofectamine), precipitate-forming agents (such as calcium phosphate),DMSO, polyethylene glycol, viral coats that package the nucleic acids,and/or so on.

[0234] Modulators may be miscellaneous chemical materials and/orbiological entities. Miscellaneous chemical modulators may be ions (suchas calcium, sodium, potassium, lithium, hydrogen (pH), chloride,fluoride, iodide, etc.), dissolved gases (NO, CO₂, O₂, etc.),carbohydrates, lipids, organics, polymers, etc. In some embodiments,biological modulators may be exposed to cells, for example, to infectcells, to measure cell-cell interactions, etc. Biological modulators mayinclude any cells, viruses, or organelles, as described above in SectionIII.

[0235] Reagents may be detection/assay reagents. Detection/assayreagents generally comprise any reagents that are contacted withparticles to facilitate processing particles (or particle components)for detection of a preexisting or newly created aspect of the particles(or components). Detection/assay reagents may include dyes, enzymes,substrates, cofactors, and/or SBP members (see Table 1 of Section VIabove), among others. Dyes, also referred to as labels, generallyinclude any optically detectable reagent. Suitable dyes may beluminophores, fluorophores, chromogens, chromophores, and/or the like.Such dyes may be conjugated to, or may be, SBP members; may act asenzyme substrates; may inherently label cells or cell structures (e.g.,DNA dyes, membrane dyes, trafficking dyes, etc.); may act as indicatordyes (such as calcium indicators, pH indicators, etc.); and/or the like.Enzymes may operate in particle assays by incorporating dyes intoproducts and/or by producing a product that may be detected subsequentlywith dyes, among others. Suitable enzymes may include polymerases (RNAand/or DNA), heat-stable polymerases (such as Taq, VENT, etc.),peroxidases (such as HRP), phosphatases (such as alkaline phosphatase),kinases, methylases, ligases, proteases, galactosidases (such asbeta-galactosidase, glucuronidase., etc.), transferases (such aschloramphenicol acetyltransferase), oxidoreductases (such asluciferase), and/or nucleases (such as DNAses, RNAses, etc.), amongothers. SBP members, such as antibodies, digoxigenin, nucleic acids,etc., may be directly conjugated to dyes, enzymes, and/or other SBPmembers; may be noncovalently bound to dyes and/or enzymes (eitherpre-bound or bound in an additional exposure step); and/or so on.Further aspects of detection/assay reagents, including the types ofassays in which these reagents may be used, are described below inSection XII.

[0236] Fluid-Mediated Mechanisms

[0237] Treatment mechanisms may use fluid-mediated mechanisms to exposeparticles to reagents. The reagents may be brought to the particles, forexample, when the particles are retained, or the particles may bebrought to the reagents, for example, when the reagents are present (andoptionally retained) in specific portions of fluid networks.

[0238] Fluid-mediated mechanisms may be flow-based, field-based, and/orpassive, among others. Flow-based treatment mechanisms may operate byfluid flow, mediated, for example, by gravity flow or active flow(pumping), to carry reagents to particles, or vice versa. In someembodiments, the flow-based treatment mechanisms may operate byregulated transverse (side-to-side) positioning, as describedabove/below in Sections V and XIII, to precisely regulate exposure ofreagents (or particles) to particles (or reagents). By contrast,field-based mechanisms may combine particles and reagents by movingreagents (or particles) with electric fields. The electric fields mayproduce any suitable electrokinetic effects, such as electrophoresis,dielectrophoresis, electroosmosis, etc. Alternatively, or in addition,reagents may be combined with particles by diffusion of the reagents.

[0239] Non-Flow-Mediated Mechanisms

[0240] Particles in microfluidic systems may be exposed to physicalmodulators/conditions using non-fluid-mediated mechanisms. However,these “non-fluid-mediated” mechanisms may use properties of fluid toassist in their operation, such as transfer of thermal energy orpressure to particles via fluid. The physical modulators/conditions maybe applied to particles from sources that are external and/or internalto the microfluidic systems. Exemplary physical modulators/conditionsmay include thermal energy (heat), radiation (light), radiation(particle), an electric field, a magnetic field, pressure (includingsound), a gravitational field, etc.

[0241] Treatment Targets

[0242] Treatment mechanisms may act on any suitable particles, includingany of the particles described above in Section III. The particles maybe intact, permeabilized, and/or lysed. Accordingly, treatmentmechanisms may act on released cell components. Particles may be treatedin arrays, either serially, for example, using a shared treatmentmechanism, and/or in parallel, for example, using distinct and/or sharedtreatment mechanisms.

[0243] Further aspects of treatment mechanisms are described above inSection V (positioning reagents/fluid/particles) and below in SectionXIII.

[0244] Measurement Mechanisms

[0245] Overview

[0246] Particles manipulated by a microfluidic system may be analyzed byone or more measurement mechanisms at one or more measurement sites. Themeasurement mechanisms generally comprise any suitable apparatus ormethod for detecting a preselected particle or particle characteristic(provided, for example, by the particle, a particle component, and/or anassay product, among others). The measurement sites generally compriseany suitable particle position or positions at which a measurement isperformed, internal and/or external to the system.

[0247] Detection Methods

[0248] The measurement mechanism may employ any suitable detectionmethod to analyze a sample, qualitatively and/or quantitatively.Suitable detection methods may include spectroscopic methods, electricalmethods, hydrodynamic methods, imaging methods, and/or biologicalmethods, among others, especially those adapted or adaptable to theanalysis of particles. These methods may involve detection of single ormultiple values, time-dependent or time-independent (e.g., steady-stateor endpoint) values, and/or averaged or (temporally and/or spatially)distributed values, among others. These methods may measure and/oroutput analog and/or digital values.

[0249] Spectroscopic methods generally may include detection of anyproperty of light (or a wavelike particle), particularly properties thatare changed via interaction with a sample. Suitable spectroscopicmethods may include absorption, luminescence (includingphotoluminescence, chemiluminescence, and electrochemiluminescence),magnetic resonance (including nuclear and electron spin resonance),scattering (including light scattering, electron scattering, and neutronscattering), diffraction, circular dichroism, and optical rotation,among others. Suitable photoluminescence methods may includefluorescence intensity (FLINT), fluorescence polarization (FP),fluorescence resonance energy transfer (FRET), fluorescence lifetime(FLT), total internal reflection fluorescence (TIRF), fluorescencecorrelation spectroscopy (FCS), fluorescence recovery afterphotobleaching (FRAP), fluorescence activated cell sorting (FACS), andtheir phosphorescence and other analogs, among others.

[0250] Electrical methods generally may include detection of anyelectrical parameter. Suitable electrical parameters may includecurrent, voltage, resistance, capacitance, and/or power, among others.

[0251] Hydrodynamic methods generally may include detection ofinteractions between a particle (or a component or derivative thereof)and its neighbors (e.g., other particles), the solvent (including anymatrix), and/or the microfluidic system, among others, and may be usedto characterize molecular size and/or shape, or to separate a sampleinto its components. Suitable hydrodynamic methods may includechromatography, sedimentation, viscometry, and electrophoresis, amongothers.

[0252] Imaging methods generally may include detection of spatiallydistributed signals, typically for visualizing a sample or itscomponents, including optical microscopy and electron microscopy, amongothers.

[0253] Biological methods generally may include detection of somebiological activity that is conducted, mediated, and/or influenced bythe particle, typically using another method, as described above.Suitable biological methods are described below in detail in SectionXII.

[0254] Detection Sites

[0255] The measurement mechanism may be used to detect particles and/orparticle characteristics at any suitable detection site, internal and/orexternal to the microfluidic system. 1

[0256] Suitable internal detection sites may include any site(s) in oron a microfluidic system (a chip). These sites may include channels,chambers, and/or traps, and portions thereof. Particles or particlecharacteristics may be detected while the particles (or releasedcomponents/assay products) are stationary or moving. Stationaryparticles may be encountered following particle retention, for example,cells growing in a cell chamber. Moving particles may be encounteredbefore and/or after particle retention, or upon confinement to a region.In particular, particles may be moved past a detection site by anysuitable positioning mechanism, for example, by fluid flow (flow-baseddetection).

[0257] Suitable external detection sites may include any site(s) awayfrom or independent of a microfluidic system. External detection sitesmay be used to detect a particle or particle characteristic afterremoval of particles (or particle components) from a microfluidicsystem. These external sites may be used instead of and/or in additionto internal sites, allowing particles (or particle components) to befurther manipulated and/or detected. These further manipulations and/ordetection methods may overlap with, but preferably complement, themanipulations and/or methods performed in the microfluidic system,including mass spectrometry, electrophoresis, centrifugation, PCR,introduction into an organism, use in clinical treatment, and/or cellculture, among others.

[0258] Detected Characteristics

[0259] The measurement method may detect and/or monitor any suitablecharacteristic of a particle, directly and/or indirectly (e.g., via areporter molecule). Suitable characteristics may include particleidentity, number, concentration, position (absolute or relative),composition, structure, sequence, and/or activity among others. Thedetected characteristics may include molecular or supramolecularcharacteristics, such as the presence/absence, concentration,localization, structure/modification, conformation, morphology,activity, number, and/or movement of DNA, RNA, protein, enzyme, lipid,carbohydrate, ions, metabolites, organelles, added reagent (binding),and/or complexes thereof, among others. The detected characteristicsalso may include cellular characteristics, such as any suitable cellulargenotype or phenotype, including morphology, growth, apoptosis,necrosis, lysis, alive/dead, position in the cell cycle, activity of asignaling pathway, differentiation, transcriptional activity, substrateattachment, cell-cell interaction, translational activity, replicationactivity, transformation, heat shock response, motility, spreading,membrane integrity, and/or neurite outgrowth, among others.

[0260] Further aspects of detected characteristics and their use inparticle assays are described below in Sections XII and XIII.

[0261] Release Mechanisms

[0262] Overview

[0263] A microfluidic system may include any suitable number of particlerelease mechanisms. A release mechanism generally comprises anymechanism(s) for allowing a retained particle to move away from apreselected site/area at which it is retained, including removing,overcoming, and/or rendering ineffective the retention mechanism(s) thatretains the particle. Release mechanisms that are suitable may beselected based, at least partially, on the retaining force. Afterrelease, particles (or particle components) may have any suitabledestination.

[0264] Removing the Retaining Force

[0265] A release mechanism may operate by removing the retaining force.Accordingly, particles that are retained by a specific mechanism may bereleased by terminating that mechanism. For example, particles retainedby a chemical interaction/bond may be released by cleaving the bond,such as with a protease(s) (e.g., trypsin), or otherwise disrupting theinteraction, such as with altered ionic conditions (e.g., with EDTA) orpH, or with an excess of a SBP member. Similarly, particles retained bya physical barrier, such as a closed valve, may be released bymoving/removing the barrier. Furthermore, particles retained by fluidflow, a vacuum, light, an electrical field, a magnetic field, and/or acentrifugal force may be released by removing/redirecting thecorresponding flow, force, field, etc.

[0266] Overcoming the Retaining Force

[0267] A release mechanism may operate by overcoming a retaining forcewith a greater force. Accordingly, particles may be released by anypositioning mechanism(s) that applies a force greater than the retainingforce. For example, retained particles may be released by a releasingflow. The releasing flow may be an increased flow rate in the directionof bulk fluid flow, for example, when a particle is weakly retained(such as by gravity/friction, or weak chemical interactions).Alternatively, the releasing flow may act counter to a retaining flow,for example orthogonal or opposite to the retaining flow. For example,the releasing flow may reposition particles to be out of contact with aretaining physical barrier (see Example 7). Alternatively, or inaddition, retained particles may be released by any other suitablepositioning mechanism(s), as described above in Section V, that iscapable of generating sufficient force.

[0268] Rendering Ineffective the Retaining Force

[0269] A release mechanism may operate by rendering ineffective theretaining force on a particle. Such a release mechanism may operate byreleasing components of the particle. For example, retained cells may belysed to release intracellular components, producing a lysate, or beadsmay be treated to release associated materials and/or tofragment/disintegrate the beads. Lysis generally includes any partial orcomplete disruption of the integrity of a cell-surface membrane, and maybe produced via temperature, a detergent, a ligand, chemical treatment,a change in ionic strength, an electric field, etc.

[0270] Destination of Released Particles/Components

[0271] Released particles and/or particle components may have anysuitable destination(s). Suitable immediate destinations may include apositioning mechanism and/or fluid surrounding the particles. Afterrelease, particles may be repositioned with a positioning mechanism,either nonselectively or selectively. Selective positioning may positionthe particle based on a measured characteristic. Positioning may be to asecond retention mechanism (and/or a culture chamber), to a detectionmechanism (such as a flow-based mechanism), and/or to an outputmechanism. Fluid surrounding the particles may be a suitable destinationfor particle components (such as cells lysates and/or bead components)to be contacted with detection/assay reagents. Alternatively, celllysates and/or bead components may be repositioned as with intactparticles.

[0272] Further aspects of release mechanisms and destinations ofreleased particles/components are described below in Section XIII.

[0273] Output Mechanisms

[0274] Microfluidic systems may include one or more output mechanismsthat interface with the microfluidic network(s). An output mechanismgenerally comprises any suitable mechanism for outputting material(s)(e.g., fluid, particles, and/or reagents) from a microfluidic system, orportions thereof, including selective and/or bulk mechanisms. The outputmechanism may direct outputted material to any suitable location, suchas an internal and/or external sink. A sink generally comprises anyreceptacle or other site for receiving outputted materials, for disposal(e.g., a waste site) or for further study or manipulation (e.g., acollection site). The outputting of materials from the microfluidicssystem may be facilitated and/or regulated using any suitablefacilitating mechanism, such as sources of internal pressure and/orexternal vacuum. The output mechanism may include a selection mechanism,such as a filter, that selects outputted materials based on somecriterion, such as whether the material is a particle or a fluid.

[0275] Cell Culture Mechanisms

[0276] Overview

[0277] Cells may be cultured using a cell culture mechanism inmicrofluidic systems. The cell culture mechanism generally comprises anysuitable mechanism for growing cells, including maintenance and/orpropagation. Suitable cells are described above in Section III.

[0278] Structural Matters

[0279] A cell culture mechanism of a microfluidic system may include oneor more culture chambers in which to culture cells. Culture chambers mayhave any suitable size, shape, composition, and/or relationship to otheraspects of microfluidic systems, based on the number of cells to becultured, size of cells, assays to performed on the cells, and/or growthcharacteristics of the cells, among others. The size of a culturechamber may be only large enough to hold one cell, several cells or more(2 to 50), or many cells (50 to 1000 or more) of a given cell size.Accordingly, culture chambers may be defined by a selected portion of apassage, an entire passage, or a set of passages. In some embodiments,culture chambers may be formed by substantially enlarged channels.Culture chambers may have any suitable height that allows cells ofinterest to enter the chamber. This height may be greater than, lessthan, and/or equal to other portions of the microfluidic network. Someor all of the surfaces of a culture chamber, such as the walls, roof,and/or substrate, may be treated or modified to facilitate aspects ofcell culture, particularly specific or nonspecific cell attachment, cellsurvival, cell growth, and/or cell differentiation (or lack thereof),among others. Suitable methods of passage treatment and treatment agentsare described above in Section VI, relative to chemical retentionmechanisms.

[0280] Culture Conditions

[0281] The cell culture mechanism may culture cells under any suitableenvironmental conditions using any appropriate environmental controlmechanisms. Suitable environmental conditions may include a desired gascomposition, temperature, rate and frequency of media exchange, and/orthe like. Environmental control mechanisms may operate internal and/orexternal to a microfluidic system. Internal mechanisms may includeon-board heaters, gas conduits, and/or media reservoirs. Externalmechanisms may include an atmosphere- and/or temperature-controlledincubator/heat source, and/or a media source external to the system. Anatmosphere-controlled incubator may be more suitable when the system isat least partially formed of a gas-permeable material, such as PDMS.Media, including gas-conditioned media, may be introduced from anexternal source by any suitable input mechanism, including manualpipetting, automated pipetting, noncontact spitting, etc. In someembodiments, the chip may be preincubated with media, which may then bediscarded, prior to the introduction of cells and/or other biologicalmaterials.

[0282] Further aspects of cell culture mechanisms, culture chambers, andculture conditions are described below in Example 10, and the materialslisted in Cross-References, particularly R. Ian Freshney, Culture ofAnimal Cells: A Manual of Basic Technique (4^(th) ed. 2000), which isincorporated herein by reference.

[0283] Particle-Based Manipulations

[0284] Overview

[0285] Microfluidic systems are used for particle manipulations.Particle manipulations generally comprise any suitable sequence ofunitary operations, for performing a desired function or assay. Unitaryoperations may be performed by each of the mechanisms described above inSections IV to X, among others.

[0286] Exemplary Sequences of Operations

[0287]FIG. 1 shows an exemplary method 100 for microfluidic manipulationand analysis of particles with systems of the invention. Each step ofmethod 100 may be repeated any suitable number of times and in anyappropriate order, as described below, based on the application.Exemplary sequences of steps are indicated by arrows.

[0288] Particles typically are initially inputted in an input step,shown at 101. Particle input introduces particles to a microfluidicsystem and may be mediated by any of the input mechanisms describedabove in Section IV.

[0289] Particles next are typically positioned, shown at 102.Positioning moves particles to selected positions along passages(longitudinal positioning), and/or to selected positions along one ormore axes generally orthogonal to the long axis (transversepositioning). Suitable positioning mechanisms that mediate one or bothof these particle movements are described above in Section V.

[0290] Particle positioning may lead to one of two paths, shown at 103and 104. Path 103 leads to particle output, shown at 105. Particleoutput may be mediated by one of the output mechanisms described abovein Section X, and may be used to discard, collect, and/or transferparticles for further analysis, among others. Path 104 leads to one ormore of three operations, particle retention 106, particle treatment107, and/or particle measurement/detection 108. These operations may beconducted in any suitable order, for any desired number of times.Particle retention mechanisms, treatment mechanisms, and measurementmechanisms are described above in Sections VI, VII, and VIII,respectively.

[0291] The steps of treating and/or measuring particles may be carriedout with or without particle retention. Accordingly, the steps oftreating and/or measuring particles may be followed directly byadditional positioning 102, or first may use a release step, shown at109, if particles have been retained. Suitable release mechanisms aredescribed above in Section IX. Alternatively, microfluidic systems maybe discarded before particle release, additional positioning, and/oroutput.

[0292] Particles that have returned to the positioning step afterentering path 104 may be manipulated further. Some or all of theseparticles may be repositioned to path 103 to be outputted 105.Alternatively, or in addition, some or all of these particles may bedirected back to path 104 to be further treated, retained, and/ormeasured. Therefore, method 100 enables any suitable sequence ofparticle manipulations and analyses at one or plural positions within amicrofluidic system.

[0293] Exemplary sequences of operations may be illustrated further asfollows. For the following discussion, the operations performed by thesteps of method 100 are abbreviated with the following single underlinedletters: Input, Position, Retain, Treat, Measure, rElease, and Output.

[0294] A basic manipulation of microfluidic analyses is IP. Thissequence of steps may lead to output (IPO) or to (path 104), resultingin the basic retention sequence IPR, flow-based measurement, IPM, orflow-based treatment, IPT.

[0295] Retained particles may be subjected to any suitable additionalsteps. The particles may be treated (IPRT), measured (IPRM), repeatedlymeasured over time (IPRMMM . . . ), treated and then measured (IPRTM),or repeatedly treated and measured (IPRTMTMTM . . . ). Retainedparticles may be released (IPR . . . E) after optional treatment and/ormeasurement. Released particles may be repositioned and then outputted(IPR . . . EPO); measured during flow (IPR . . . EPM); treated (IPR . .. EPT); treated and measured (IPR . . . EPTM); retained and treated (IPR. . . EPRT); retained, treated, and measured, (IPR . . . EOPRTM); and/orso on.

[0296] Cell-Based Assays/Methods

[0297] The microfluidic systems of the invention may be used for anysuitable cell assays or methods, including any combinations of cells,cell selection(s) (by selective retention), treatment(s), and/ormeasurement(s), as described above in Sections III, VI, VII, and VIII,respectively.

[0298] The cell assays may characterize cells, either with or withoutaddition of a modulator. Cell assays may measure cell genotypes,phenotypes, and/or interactions with modulators. These assays maycharacterize individual cells and/or cell populations/groups of anysuitable size. Cells may be characterized in the absence of an addedmodulator to define one or more characteristics of the cells themselves.Alternatively, or in addition, cell may be characterized in the presenceof an added modulator to measure interaction(s) between the cells andthe modulator. Moreover, cells may be exposed to a selectedconcentration of a reagent, or a plurality of concentrations of areagent. In other embodiments, cells are exposed to a gradient ofconcentrations of reagent to determine whether such cells will beattracted or repelled by increasing amounts of such reagent.

[0299] In other embodiments, a quantity of cells may be measured out byfirst filling a measuring chamber having at least one inlet, the inlethaving at least one valve, where the valve is opened, cells areintroduced into the chamber, preferably by blind filling a dead-endchamber, or by opening up an outlet valve to an outlet in communicationwith the chamber, the outlet having a retention mechanism for preventingthe cells from exiting the chamber. The measure amount of cells is thendisplaced to a culturing region for culturing.

[0300] In other embodiments, a first type of cell is grown in fluidcommunication with a second type of cell, wherein the first type of cellis affected by the presence of the second type of cell, preferably as aco-culture or feeder type relationship. The cells of the first type andthe cells of the second type are kept separate from each other by aretention mechanism, although fluid, preferably liquid, is permitted tobe in joint contact with each type of cell so that sub-cellular orbiochemical materials may be exchanged between cell types.

[0301] Genotypic Assays

[0302] Genotypic assays may be conducted on cells in microfluidicsystems to measure the genetic constitution of cells. The genotypicassays may be conducted on any suitable cell or cell populations, forexample, patient samples, prenatal samples (such as embryonic, fetal,chorionic villi, etc.), experimentally manipulated cells (such astransgenic cells), and/or so on. Such genotypic aspects may include copynumber (such as duplication, deletion, amplification, and/or the like)and/or structure (such as rearrangement, fusion, number of repeats (suchas dinucleotide, triplet repeats, telomeric repeats, etc.), mutation,gene/pseudogene, specific allele, presence/absence/identity/frequency ofsingle nucleotide polymorphisms, integration site, chromosomal/episomal,and/or the like) of a nuclear and/or mitochondrial gene(s), genomicregion(s), and/or chromosomal region (s) (such as telomeres,centromeres, repetitive sequences, etc.). Methods for genotypic assaysmay include nucleic acid hybridization in situ (on intact cells/nuclei)or with DNA released from cells, for example, by lysing the cells.Nucleic acid hybridization with nucleic acids may be carried out with adye-labeled probe, a probe labeled with a specific binding pair (seeSection VI), a stem-loop probe carrying an energy transfer pair (such asa “molecular beacon”), and/or with a probe that is labeled enzymaticallyafter hybridization (such as by primer extension with a polymerase,modification with terminal transferase, etc). Alternatively, or inaddition, methods for genotypic assays may include polymerase-mediatedamplification of nucleic acids, for example, by thermal cycling (PCR) orby isothermal strand-displacement methods. In some embodiments,genotypic assays may use electrophoresis to assist in analysis ofnucleic acids. Related gene-based assays may measure other aspects ofgene regions, genes, chromosomal regions, whole chromosomes, or genomes,using similar assay methods, and suitable probes or DNA dyes (such aspropidium iodide, Hoechst, etc.). These other aspects may include totalDNA content (for example 2N, 4N, 8N, etc., to measure diploid,tetraploid, or polyploid genotypes and/or cell cycle distribution),number or position of specific chromosomes, and/or position of specificgenes (such as adjacent the nuclear membrane, another nuclear structure,and so on).

[0303] Phenotypic Assays

[0304] Phenotypic assays may be conducted to characterize cells inmicrofluidic systems, based on genetic makeup and/or environmentalinfluences, such as presence of modulators. These assays may measure anymolecular or cellular aspect of whole cells, cellular organelles, and/orendogenous (native) or exogenous (foreign) cell constituents/components.

[0305] Aspects of a whole cell or whole cell population may includenumber, size, density, shape, differentiation state, spreading,motility, translational activity, transcriptional activity, mitoticactivity, replicational activity, transformation, status of one or moresignaling pathways, presence/absence of processes, intact/lysed,live/dead, frequency/extent of apoptosis or necrosis,presence/absence/efficiency of attachment to a substrate (or to apassage), growth rate, cell cycle distribution, ability to repair DNA,response to heat shock, nature and/or frequency of cell-cell contacts,etc.

[0306] Aspects of cell organelles may include number, size, shape,distribution, activity, etc. of a cell's (or cell population's) nuclei,cell-surface membrane, lysosomes, mitochondria, Golgi apparatus,endoplasmic reticulum, peroxisomes, nuclear membrane, endosomes,secretory granules, cytoskeleton, axons, and/or neurites, among others.

[0307] Aspects of cell constituents/components may includepresence/absence or level, localization, movement, activity,modification, structure, etc. of any nucleic acid(s), polypeptide(s),carbohydrate(s), lipid(s), ion(s), small molecule, hormone, metabolite,and/or a complex(es) thereof, among others. Presence/absence or levelmay be measured relative to other cells or cell populations, forexample, with and without modulator. Localization may be relative to thewhole cell or individual cell organelles or components. For example,localization may be cytoplasmic, nuclear, membrane-associated,cell-surface-associated, extracellular, mitochondrial, endosomal,lysosomal, peroxisomal, and/or so on. Exemplary cytoplasmic/nuclearlocalization may include transcription factors that translocate betweenthese two locations, such as NF-κB, NFAT, steroid receptors, nuclearhormone receptors, and/or STATs, among others. Movement may includeintracellular trafficking, such as protein targeting to specificorganelles, endocytosis, exocytosis, recycling, etc. Exemplary movementsmay include endocytosis of cell-surface receptors or associated proteins(such as GPCRs, receptor tyrosine kinases, arrestin, and/or the like),either constitutively or in response to ligand binding. Activity mayinclude functional or optical activity, such as enzyme activity,fluorescence, and/or the like, for example, mediated by kinases,phosphatases, methylases, demethylases, proteases, nucleases, lipases,reporter proteins (for example beta-galactosidase, chloramphenicolacetyltransferase, luciferase, glucuronidase, green fluorescent protein(and related derivatives), etc.), and/or so on. Modification may includethe presence/absence, position, and/or level of any suitable covalentlyattached moiety. Such modifications may include phosphorylation,methylation, ubiquitination, carboxylation, and/or farnesylation, amongothers. Structure may include primary structure, for example afterprocessing (such as cleavage or ligation), secondary structure ortertiary structure (e.g., conformation), and/or quaternary structure(such as association with partners in, on, or about cells). Methods formeasuring modifications and/or structure may include specific bindingagents (such as antibodies, etc.), in vivo or in vitro incorporation oflabeled reagents, energy transfer measurements (such as FRET), surfaceplasmon resonance, and/or enzyme fragment complementation or two-hydridassays, among others.

[0308] Nucleic acids may include genomic DNA, mitochondrial DNA, viralDNA, bacterial DNA, phage DNA, synthetic DNA, transfected DNA, reportergene DNA, etc. Alternatively, or in addition, nucleic acids may includetotal RNAs, hnRNAs, mRNAs, tRNAs, siRNAs, dsRNAs, snRNAs, ribozymes,structural RNAs, viral RNAs, bacterial RNAs, gene-specific RNAs,reporter RNAs (expressed from reporter genes), and/or the like. Methodsfor assaying nucleic acids may include any of the techniques listedabove under genotypic assays. In addition, methods for assaying nucleicacids may include ribonuclease protection assays.

[0309] Polypeptides may include any proteins, peptides, glycoproteins,proteolipids, etc. Exemplary polypeptides include receptors, ligands,enzymes, transcription factors, transcription cofactors, ribosomalcomponents, regulatory proteins, cytoskeletal proteins, structuralproteins, channels, transporters, reporter proteins (such as thoselisted above which are expressed from reporter genes), and/or the like.Methods for measuring polypeptides may include enzymatic assays and/oruse of specific binding members (such as antibodies, lectins, etc.),among others. Specific binding members are described in Section VI.

[0310] Carbohydrates, lipids, ions, small molecules, and/or hormones mayinclude any compounds, polymers, or complexes. For example,carbohydrates may include simple sugars, di- and polysaccharides,glycolipids, glycoproteins, proteoglycans, etc. Lipids may includecholesterol and/or inositol lipids (e.g., phosphoinositides), amongothers; ions may include calcium, sodium, chloride, potassium, iron,zinc, hydrogen, magnesium, heavy metals, and/or manganese, among other;small molecules and/or hormones may include metabolites, and/or secondmessengers (such as cAMP or cGMP, among others), and/or the like.Concentration gradients and/or movement of ions may provide electricalmeasurements, for example, by patch-clamp analysis, as described inExamples 11 and 12.

[0311] Interaction Assays

[0312] Interaction generally comprises any specific binding of amodulator to a cell or population of cells, or any detectable change ina cell characteristic in response to the modulator. Specific binding isany binding that is predominantly to a given partner(s) that is in, on,or about the cell(s). Specific binding may have a binding coefficientwith the given partner of about 10⁻³ M and lower, with preferredspecific binding coefficients of about 10⁻⁴ M, 10 ⁻⁶ M, or 10 ⁻⁸ M andlower. Alternatively, interaction may be any change in a phenotypic orgenotypic characteristic, as described above, in response to themodulator.

[0313] Interaction assays may be performed using any suitablemeasurement method. For example, the modulator may be labeled, such aswith an optically detectable dye, and may be labeled secondarily afterinteraction with cells. Binding of the dye to the cell or cells thus maybe quantified. Alternatively, or in addition, the cell may be treated orotherwise processed to enable measurement of a phenotypic characteristicproduced by modulator contact, as detailed above and in Section VIII.

[0314] Cells and/or cell populations may be screened with libraries ofmodulators to identify interacting modulators and/or modulators withdesired interaction capabilities, such as a desired phenotypic effect(such as reporter gene response, change in expression level of a nativegene/protein, electrophysiological effect, etc.) and/or coefficient ofbinding. A library generally comprises a set of two or more members(modulators) that share a common characteristic, such as structure orfunction. Accordingly, a library may include two or more smallmolecules, two or more nucleic acids, two or more viruses, two or morephages, two or more different types of cells, two or more peptides,and/or two or more proteins, among others.

[0315] Signal Transduction Assays

[0316] Microfluidic assays of cells and/or populations may measureactivity of signal transduction pathways. The activity may be measuredrelative to an arbitrary level of activity, relative to other cellsand/or populations (see below), and/or as a measure of modulatorinteraction with cells (see above).

[0317] Signal transduction pathways generally comprise any flow ofinformation in a cell. In many cases, signal transduction pathwaystransfer extracellular information, in the form of a ligand(s) or othermodulator(s), through the membrane, to produce an intracellular signal.The extracellular information may act, at least partially, by triggeringevents at or near the membrane by binding to a cell-surface receptor,such as a G Protein-Coupled Receptor (GPCR), a channel-coupled receptor,a receptor tyrosine kinase, a receptor serine/threonine kinase, and/or areceptor phosphatase, among others. These events may include changes inchannel activity, receptor clustering, receptor endocytosis, receptorenzyme activity (e.g., kinase activity), and/or second messengerproduction (e.g., cAMP, cGMP, diacylglcyerol, phosphatidylinositol,etc.). Such events may lead to a cascade of regulatory events, such asphosphorylation/dephosphorylation, complex formation, degradation,and/or so on, which may result, ultimately, in altered gene expression.In other cases, modulators pass through the membrane and directly bindto intracellular receptors, for example with nuclear receptors (such assteroid receptors (GR, ER, PR, MR, etc.), retinoid receptors, retinoid Xreceptor (RXRs), thyroid hormone receptors, peroxisomeproliferation-activating receptors (PPARs), and/or xenobiotic receptors,among others). Therefore, any suitable aspect of this flow ofinformation may be measured to monitor a particular signal transductionpathway.

[0318] The activity measured may be based at least partially, on thetype of signal transduction pathway being assayed. Accordingly, signaltransduction assays may measure ligand binding; receptorinternalization; changes in membrane currents; association of receptorwith another factor, such as arrestin, a small G-like protein such asrac, or rho, and/or the like; calcium levels; activity of a kinase, suchas protein kinase A, protein kinase C, CaM kinase, myosin light chainkinase, cyclin dependent kinases, P13-kinase, etc.; cAMP levels;phosholipase C activity; subcellular distribution of proteins, forexample, NF-κB, nuclear receptors, and/or STATs, among others.Alternatively, or in addition, signal transduction assays may measureexpression of native target genes and/or foreign reporter genes thatreport activity of a signal transduction pathway(s). Expression may bemeasured as absence/presence or level of RNA, protein, metabolite, orenzyme activity, among others, as described above.

[0319] Comparison of Cells and/or Cell Populations

[0320] Cell-based assays may be used to compare genotypic, phenotypic,and/or modulator interaction of cells and/or populations of cells. Thecells and/or populations may be compared in distinct microfluidicsystems or within the same microfluidic system. Comparison in the samemicrofluidic system may be conducted in parallel using a side-by-sideconfiguration, as exemplified by Example 3, in parallel at isolatedsites, as exemplified by Example 4, and/or in series, as exemplified byExample 5.

[0321] Single-Cell Assays

[0322] Microfluidic systems may be used to perform single-cell assays,which generally comprise any assays that are preferably or necessarilyperformed on one cell at a time. Examples of single cell assays includepatch-clamp analysis, single-cell PCR, single-cell fluorescence in situhybridization (FISH), subcellular distribution of a protein, and/ordifferentiation assays (conversion to distinct cell types). In somecases, single-cell assays may be performed on a retained group of two ormore cells, by measuring an individual characteristic of one member ofthe group. In other cases, single-cell assays may require retention of asingle cell, for example, when the cell is lysed before the assay.

[0323] Sorting/Selection

[0324] Microfluidic systems may be used to sort or select single cellsand/or cell populations. The sorted/selected cells or populations may beselected by stochastic mechanisms (see Example 2), size, density,magnetic properties, cell-surface properties (that is, ability to adhereto a substrate), growth and/or survival capabilities, and/or based on ameasured characteristic of the cells or populations (such as response toa ligand, specific phenotype, and/or the like). Cells and/or populationsmay be sorted more than once during manipulation and/or analysis in amicrofluidic system. In particular, heterogeneous populations of cells,such as blood samples or clinical biopsies, partially transfected ordifferentiated cell populations, disaggregated tissues, natural samples,forensic samples, etc. may be sorted/selected. Additional aspects ofcell sorting and suitable cells and cell populations are described abovein Section III and below in Examples 9, 15, 23, and 26.

[0325] Storage/Maintenance

[0326] Microfluidic systems may perform storage and/or maintenancefunctions for cells. Accordingly, cells may be introduced intomicrofluidic systems and cultured for prolonged periods of time, such aslonger than one week, one month, three months, and/or one year. Usingmicrofluidic systems for storage and/or maintenance of cells may consumesmaller amounts of media and space, and may maintain cells in a moreviable state than other storage/maintenance methods. Additional aspectsof storing and maintaining cells in microfluidic systems are included inSection XI above and Example 10 below.

[0327] Assays/Methods with Other Particles

[0328] Microfluidic systems may be used for any suitable virally based,organelle-based, bead-based, and/or vesicle-based assays and/or methods.These assays may measure binding (or effects) of modulators (compounds,mixtures, polymers, biomolecules, cells, etc.) to one or more materials(compounds, polymers, mixtures, cells, etc.) present in/on, orassociated with, any of these other particles. Alternatively, or inaddition, these assays may measure changes in activity (e.g., enzymeactivity), an optical property (e.g., chemiluminescence, fluorescence,or absorbance, among others), and/or a conformational change induced byinteraction.

[0329] In some embodiments, beads may include detectable codes. Suchcodes may be imparted by one or more materials having detectableproperties, such as optical properties (e.g., spectrum, intensity, andor degree of fluorescence excitation/emission, absorbance, reflectance,refractive index, etc.). The one or more materials may providenonspatial information or may have discrete spatial positions thatcontribute to coding aspects of each code. The codes may allow distinctsamples, such as cells, compounds, proteins, and/or the like, to beassociated with beads having distinct codes. The distinct samples maythen be combined, assayed together, and identified by reading the codeon each bead. Suitable assays for cell-associated beads may include anyof the cell assays described above.

[0330] Suitable protocols for performing some of the assays described inthis section are included in Joe Sambrook and David Russell, MolecularCloning: A Laboratory Manual (3^(rd) ed. 2000), which is incorporatedherein by reference.

EXAMPLES

[0331] The following examples describe selected aspects and embodimentsof the invention, including methods of fabricating, integrating, andusing microfluidic systems, and devices, and mechanisms for manipulationand analysis of particles. These examples are included for illustrationand are not intended to limit or define the entire scope of theinvention.

[0332] Many of the examples presented below include figures showingmolds, fluid layers, and/or control layers that are color-coded. Sincemolds and fluid or control layers have complementary patterns, thecolor-coded schemes generally represent both molds and fluid or controllayers, although one or the other is often designated in thecorresponding description. Throughout these examples, the colors ofmolds and/or fluidic layers have the following meanings: regions in redhave a height of approximately 20 μm, and a rectangular cross-sectionalgeometry; regions in blue have a height of about 20 μm, and asemi-circular/arcuate cross-sectional geometry; regions in turquoisehave a height of about 5 μm and a rectangular cross-sectional geometry;and regions in white are not raised from the general surface of the moldand/or form a portion of the substrate-contacting surface of a fluidlayer. The widths of these regions are generally cited in the text.

[0333] Dimensions and cross-sectional geometries presented in theseexamples are exemplary only, being designed for particles of about 8 to12 μm in diameter. Accordingly, any absolute or relative dimensions orcross-sectional geometries may be selected based the application and thesize of input particles being analyzed. Thus, the regions in red andblue may have a height of about 0.5 to 100, 1 to 75, or 2 to 50 μm.Regions in turquoise may have a height of about 0.1 to 50, 0.2 to 25, orabout 0.5 to 20 μm. In addition, these regions may have any suitablecross-sectional geometries based on the application. Furthermore,regions in red and blue may have any suitable width based on theirfunction. For example, regions in red used for particle positioning mayhave widths of at least about 2, 10, 20, or 50 μm. By contrast, regionsin red used for reagent dispensing may have smaller widths of at leastabout 0.2, 1, 2, or 5 μm. Regions in blue may have widths of at leastabout 5, 10, 20, or 50 μm.

Example 1 Cell Positioning and Retention Mechanisms

[0334] This example describes microfluidic systems for positioningand/or retaining single particles or groups of particles, based, atleast in part, on divergent flow paths; see FIGS. 2-4.

[0335] Background

[0336] There are many cell analyses that benefit from or require theprecise positioning and retention of a single cell or a small group ofcells. In particular, positioned and retained cells may be treated andobserved in real time. However, currently available mechanisms forpositioning and retaining cells are either expensive and laborintensive, or imprecise and deleterious to cells. For example,micromanipulators enable a user to select and precisely position asingle cell. However, micromanipulators are expensive, and require thatusers observe the cell throughout the micromanipulation. Hence, the usercan only position one cell at a time. At the other extreme, filtersoffer a crude, but much cheaper and faster mechanism for positioning andretaining cells. However, filters have a number of disadvantages. Forexample, they are easy to clog, difficult to control (particularly withregard to the number of retained cells), and potentially harmful toparticles such as cells due to the pressure drop across the filter.Therefore, there is a need for cell positioning and retention systemsthat are economical, guided automatically without optical monitoring,and/or able to gently manipulate cells without substantially damagingthem.

[0337] Description

[0338] This example describes mechanisms for positioning and/orretaining particles such as cells and/or beads without requiring opticalmonitoring. Once retained, the particles may be analyzed by any suitablemethod, including optical and electrical methods, among others. Thedescribed mechanisms use a microfluidic flow path that diverges to forma quasi-stagnant fluidic region at the position of divergence. Particlesentering this quasi-stagnant fluidic region from a microfluidic streamexperience a reduction in velocity, which may be exploited to effecttheir “soft landing” in a suitable retention structure or trap.Accordingly, the retained particles are more likely to be undamaged andsuitable for subsequent analyses.

[0339] Embodiment 1

[0340]FIG. 2A shows a system 110 for microfluidic manipulation and/oranalysis of particles, in accordance with aspects of the invention.System 10 includes (1) an input reservoir 112, (2) a microfluidicnetwork ll4 having three fluidic channels 116, 118, 120, and (3) twooutput or waste reservoirs 122, 124. Particles are loaded, generally insuspension, into input reservoir 112. The loaded particles may enternetwork 114 in response to net fluid flow, shown as flow streams 126,128, 130, between the input and waste reservoirs. The net fluid flow maybe determined by active and/or passive flow, mediated, for example, bypumping and/or gravity, respectively.

[0341] The bifurcation of fluid flow stream 126 into flow streams 128,130 creates a positioning mechanism 132. This positioning mechanism usesa reduced-velocity flow stream 134, shown as a dotted arrow, to gentlyposition a fraction of particles through an extension of flow stream126.

[0342] Particles may be carried by flow stream 134 into a suitableretention mechanism 136. In system 110, this retention mechanismincludes a recess 138 formed in opposing wall 140, near a terminal endof reduced-velocity flow stream 134. Recess 138 may have a width anddepth that accommodates one particle or a group of two or moreparticles. Recess 138 includes retention structures 142 that blockmovement of retained particles, generally in the direction of flowstreams 128, 130. The depth of recess 138, coupled with any extension ofretention structures 142, generally away from wall 140, may determinethe number of particles retained and their associated retentionefficiency. Thus, retention mechanism 136 may effect stable or transientretention of particles. Transient retention may provide an average timeof occupancy that is suitable for treatment and/or analysis, followed bystochastic loss and replacement of a particle or particles by otherparticles entering along reduced-velocity flow stream 134.

[0343] Particles retained by retention mechanism 136 may be treatedand/or analyzed. In some embodiments, retained particles are analyzedelectrically, for example, using an electrode 143. Alternatively, or inaddition, retained particles may be treated and/or analyzed and thenremoved by a suitable release mechanism 144. For example, in system 110,the release mechanism applies a dislodging pressure on retained cellsthat opposes flow stream 134. Release mechanisms are described furtherin Section IX above and in Examples 7 and 26 below.

[0344] Embodiment 2

[0345]FIG. 2B shows another system 110′ for microfluidic manipulationand/or analysis of particles, in accordance with aspects of theinvention. The operational principles for system 110′ of FIG. 2B aresimilar to those for system 110 of FIG. 2A. However, channels 118′ and120′ diverge less than 90° from channel 116′ in system 110′, in contrastto their orthogonally directed counterparts in system 110. Consequently,a greater fraction of particles may be positioned in flow stream 134′ insystem 110′ than in flow stream 134 in system 110, but a greaterdislodging force also may be present. In other embodiments, the outputchannels may have any suitable angles of divergence, including greaterthan 90°, and/or they may have unequal angles of divergence. The anglesof divergence and any asymmetry in the two fluid paths may be alterableto select the number of particles trapped and/or retained and theirpositions within the trap.

[0346] Embodiment 3

[0347]FIG. 3 shows yet another system 170 for microfluidic manipulationand/or analysis of particles, in accordance with aspects of theinvention. System 170 includes (1) a fluidic network 172 of channels174, 176, 178 and (2) a retention mechanism or trap 180. A flow stream181 brings input sample and fluid to a T-junction 182, at which stream181 is divided into orthogonally directed, primary flow-streams 184,186. As in systems 110, 110′ of FIGS. 2A and 2B, a reduced velocity,positioning flow-stream 188 extends from stream 181, between primarystreams 184, 186, toward opposing wall 188. However, unlike systems 110and 110′, system 170 also includes partitions 192, 194 (“P” and “Q”,respectively) in the form of rectangular blocks. Partitions 192, 194divide the main channels to create secondary channels 196, 198, whichextend generally parallel to main channels 176, 178. These secondarychannels divide positioning flow-stream 188 and direct it orthogonallyin opposite directions, as shown by secondary flow streams 200, 202.Secondary flow streams transport fluid at a lower velocity than primarystreams 184, 186 because of their position within network 172.

[0348]FIG. 4 shows system 170 during particle input, after positioningand retention of a single particle 204 between partitions 192, 194 bytrap 180. Particles 206 entering network 172 may travel along flowstream 181, generally in both central and lateral positions withinchannel 174. Laterally positioned cells, such as cells 208, followprimary flow streams 184, 186 along channels 176, 178. In contrast,centrally positioned cells, such as cells 210, follow positioning stream188 toward a slot or gap 212 between partitions 192, 194. In thisembodiment, gap 212 is slightly wider than the diameter of cells 206, sothat it will accept only one cell. In other embodiments, and/or forother cells, gap 212 may be wide enough to accept two or more cells.Whatever the width of gap 212, wall 190 and partitions 192, 194, form aretention site 214 at which cell 204 or cells may be stably retained.Once cell 204 is positioned at the retention site by trap 180, itspresence may tend to block or diminish fluid flow along secondarystreams 200, 202, through secondary channels 196, 198 (see FIG. 3).Accordingly, secondary streams 200, 202 have diminished capacity to drawadditional cells between partitions 192, 194. As a result, in someembodiments, trap 180 may preferentially retain only one cellautomatically, without any need for optical monitoring duringpositioning and/or retention. Thus, retention site 214 may bedimensioned based on the size of cells to be retained. For example,eukaryotic cells typically are about 2 to 10 μm in diameter, so gap 212may be slightly wider than this diameter, whereas secondary channels196, 198 may be slightly narrower than this diameter, to prevent entryof cells into these channels.

[0349] Retained cell 204 may be treated and/or analyzed using anysuitable method, such as optical and/or electrical detection of cellcharacteristics, as described above in Section VIII. This treatmentand/or analysis may be facilitated by a microchannel 216 that extendsoutward from wall 190 into chamber 218. Microchannel 216 is smaller thanthe diameter of retained cell 204 and may be used to exert positiveand/or negative pressure on the retained cell, or apply and/or measurean electrical potential and/or current across the retained cell, amongothers, as described below in Examples 11 and 12.

Example 2 Microfluidic Systems for Trapping and Perfusing Particles

[0350] This example describes microfluidic systems that position andretain single particles or sets of particles, and allow rapid, preciseperfusion of the retained particles or sets of particles with reagents;see FIGS. 5-11C.

[0351] Background

[0352] Many cell studies benefit from analysis of a population of cells.The population may provide discrete information from individual cells ofthe population and averaged information from the entire population.Accordingly, a population of cells may allow concurrent analysis ofdistinct types of cells when the population is heterogeneous, or a rangeof cell phenotypes or responses when the population is homogeneous orclonal. Therefore, studies of cells in a microfluidic environment wouldbenefit from microfluidic systems that automatically position and/orretain a set of cells at a preselected site on a microfluidic chip.Furthermore, these studies would benefit from mechanisms that allow theretained set of cells to be perfused with selected reagents, such asdrugs, test compounds, or labels, in a controllable and definablemanner.

[0353] Description

[0354] This example describes microfluidic systems that enable a user totrap multiple cells within a cell retention chamber, and perfuse thetrapped cells with reagents for controlled intervals. These systems maybe formed by any suitable method, including multilayer soft lithographyinvolving multiple layers of photoresist, for example, using moldsfabricated as described below in Example 13 and elsewhere in thisDetailed Description, and in the patent applications listed above underCross-References and incorporated herein by reference. Accordingly, insome embodiments, the cross-sectional geometry of fluidic channels mayvary between rectangular in flow channels and arcuate at the position ofvalves.

[0355] Embodiment 1

[0356] FIGS. 5-11 show a system 250 for microfluidic analysis of cellpopulations. This system is described in detail below, including (a)system description, (b) system production, (c) system operation, and (d)system protocols.

[0357] System Description

[0358]FIG. 5 shows a portion of a system 250 for microfluidic analysisof cell populations. System 250 includes a microfluidic layer 252 and acontrol layer 254. Microfluidic layer 252 forms a microfluidic network256 of interconnected channels, depicted in blue and orange. Controllayer 254 is positioned over, and abutting, the microfluidic layer, andincludes valves and pumps (see also FIG. 8), depicted in purple.Exemplary dimensions presented below for system 250 are based on celldiameters of about 8 to 12 μm.

[0359] The microfluidic layer includes microfluidic channels withdistinct geometries and functions. Blue, flow channels 258 have asemi-circular or arcuate cross-sectional profile and are positionedgenerally upstream and downstream of mechanisms for cell positioning,retention, and/or treatment, which are described below. These flowchannels have cross-sectional profiles that allow the channels to beacted upon effectively by valves and pumps present in control layer 254.In this example, flow channels are about 200 μm wide and 20 μm high. Incontrast, orange, cell channels 260 have a rectangular profile. In thisexample, cell channels are about 100 μm wide and 20 μm high. Becausechannel height does not restrict lateral movement, at least to firstorder, the cells or particles can travel freely within the cell channel,following the walls or more central positions based on the particularlaminar flow stream that carries a particular cell or particle. Thus,these cell channels are used to position cells to preselected laminarflow streams and preselected regions of the microfluidic network.Perfusion channels 262, described more fully below, also are shown inorange and function to controllably perfuse retained cells. In thisparticular example, perfusion channels are about 10 μm wide.

[0360] System 250 includes an input mechanism 263, a positioningmechanism 264, a retention mechanism 266, and a perfusion mechanism 268.The positioning and retention mechanisms function together to positionand trap cells in a retention or capture chamber 270. The perfusionmechanism functions to effect delivery of reagents to the cells inretention chamber 270, typically after cell retention.

[0361] Input mechanism 263 introduces particles into the system, usingan input reservoir or well, as described below (see FIG. 8).

[0362] Positioning mechanism 264 operates to increase the probabilitythat input cells will enter the retention chamber. Mechanism 264operates through convergent flow streams that join but remain segregatedin a laminar distribution. Input flow streams 272, 274, 276 carry fluidalong flow channels 278, 280, 282, respectively. However, channel 280also may carry cells, whereas flanking channels 278, 282 generally donot. As a result, at confluence 284, flow stream 274 occupies a centralportion, flanked by flow streams 272, 276. Accordingly, the accompanyingcells are focused to a central portion of combined stream 286. In someembodiments, additional flow streams may be included, and/or cells maybe included in other flow streams, as exemplified below in Example 3.

[0363]FIGS. 6 and 7 show, respectively, corresponding actual andschematic views of the retention mechanism or trap 266 of FIG. 5. Theretention mechanism includes a partially closed retention or capturechamber 270. Chamber 270 may have a size of about 60-100 μm long, 50-100μm wide, and 20 μm high. Chamber 270 is formed by opposing channel wall288, front wall 290, side walls 292, 294, and top and bottom walls (notshown). Front wall includes an aperture 296 through which cells enterthe chamber from a reduced-velocity stream 298, extending from combinedstream 286. The reduced-velocity stream may be less damaging to cellsthat enter the chamber, increasing viability and the probability of afruitful analysis. Aperture 296 is about 5-20 μm wide and may have aheight corresponding to some or all of the channel height. Fluidentering aperture 296 as part of stream 298 may pass through side-wallchannels 300. In this example, each side wall includes three side-wallchannels 300, which have a rectangular profile about 10 μm wide and 5 μmhigh. In general, the side-wall channels are dimensioned to selectivelyretain cells or particles of interest, while allowing fluid or smallercells or particles to pass through. Thus, chamber 270 functions as afilter. However, in contrast to standard filters, only a fraction ofinput cells enter chamber 270. The fraction may be less than about 1 in10, 1 in 100 or 1 in 1,000, among others, depending on the design of theretention chamber, the speed of the input fluid stream, and the size anddensity of particles, among others.

[0364]FIG. 7 shows a focused stream of cells 302 flowing toward chamber270. Cells 302 either enter aperture 296 or are carried orthogonally bychannels 304, 306. Within chamber 270 microstreams 308 connect chamber270 with side-wall channels 300.

[0365] Perfusion mechanism 268 provides precisely controlled exposure toreagents for trapped cells in chamber 270. FIG. 5 shows the generaldesign of the perfusion mechanism. Trapped cells are selectively exposedto buffer or reagent streams carried by one of two or more perfusionchannels 310, 312. Fluid, such as media, buffer, and/or reagent, flowsthrough perfusion channels 310 and/or 312 and joins focusing bufferstream 314. During perfusion, focusing buffer stream 314 is produced byinput fluid from one or more input reservoirs “B,” described more fullybelow, flowing past chamber 270 in a single stream. Thus, the stream nois longer split as occurs during cell positioning and retention, asshown in FIG. 7. Due to laminar flow and the position of perfusionchannels 310, 312, fluid from either one of these channels enters tojoin main flow stream 314 on the side of the main flow stream nearestchamber 270. Therefore, the trapped cells are exposed to fluid fromperfusion channel 310 or 312. However, if fluid is flowing from bothperfusion channels, fluid from perfusion channel 312 shields trappedcells from fluid flowing from perfusion channel 310, such as a reagent.Accordingly, the contents of perfusion channel 312 may be referred to asa shield liquid or shield buffer. With concurrent flow from bothperfusion channels, cells may be rapidly exposed to a reagent fromperfusion channel 310 by stopping flow from channel 312. Stopping theflow of the perfusion buffer may expose cells to reagent within a veryshort time, in some cases about 150 msec after stopping flow. Therefore,cell analyses that require precise control of reagent exposure tomeasure rapid cell responses may be conducted reproducibly with therapid response times afforded by this microfluidic system.

[0366] Perfusion mechanism 268 may be modified to achieve similarperfusion or to change the exposure response time. For example, similarperfusion may be obtained by disposing perfusion channels on opposingsides of transverse channel 316, or disposing both perfusion channels onopposing wall 288. Alternatively, or in addition, the exposure time maybe increased or reduced by moving perfusion channel 310 closer to, orfarther from, main flow stream 314. Example 3 shows a perfusion channelthat empties directly into the focusing buffer stream.

[0367]FIG. 8 shows additional aspects of microfluidic system 250. Theseadditional aspects include macrofluidic reservoirs, and valves and/orpumps of the control layer that control fluid flow within themicrofluidic network.

[0368] Macrofluidic reservoirs allow system 250 to interface with themacroscopic world. Each reservoir or well functions as a fluidic inletor outlet connected directly to at least one microfluidic channel.Fluidic inlet-well A, shown at 330, provides for particle input,generally as a cell suspension. Fluidic inlet-well B, shown at 332,holds a focusing buffer, which is split into two focusing channels, 334,336, that ultimately form converging flow streams 272, 276. Fluidicoutlet-well C, shown at 338, holds output liquid, generally wasteliquid, that flows through the system. Well C accepts fluid from one orboth of fluid channels 340, 342. Fluidic inlet-wells D and E, shown at344 and 346, may hold first and second reagents for exposure to trappedcells. Fluidic inlet-well F, shown at 348, holds the shield buffer thatblocks exposure of the reagents until desired.

[0369] Control layer interfaces are numbered one through eleven. Eachinterface acts as a gas inlet to regulate opening and closing of one ormore valves. Interface seven controls cell input valve 350. Similarly,interface eight controls fluid channel 340, determining whether mainflow stream 314 bifurcates or is a single stream. Interfaces nine, ten,and eleven control valves 352, 354, 356, which regulate inflow ofreagent or shield buffer from fluidic inlets D, E, and F, respectively.Interfaces 1 through 3 and 4 through 6 control sets of values, shown at358 and 360, respectively. Valves within each set are actuated in adefined sequence to pump liquid by peristalsis from inlets B (valve set360) or D-F combined (valve set 358).

[0370] System Production

[0371] System 250 may be formed using any suitable method. In anexemplary approach, the system is formed by layering and fusingmicrofluidic layer 252, control layer 254, and a substrate layer,formed, for example, by a cover slip (not shown). Specifically, in thisapproach, the microfluidic and control layers are molded by softlithography and then fused. Next, the resulting fused multilayerstructure is bonded to the cover slip substrate layer. Finally,microfluidic channels are wetted with deionized water.

[0372] System Operation

[0373] System 250 may be used to load, position, and/or retainparticles, such as cells, using any suitable method. In an exemplaryapproach, valves 7, 9, 10, 11 are closed, and the remaining valves,including the pump valves, are opened. Wells B and F are loaded withfocusing and shield buffers, respectively, wells D and E are loaded withreagents, and well A is loaded with a cell suspension. Valve 7 is thenopened, after ensuring that waste well C is at least partly empty,enabling cells to flow towards well C. At this point, no liquid flowsfrom wells D, E, and F. Buffer flows from well B to well C, and cellsflow from well A to well C. The cells flowing out of well A are focusedin the center of combined flow stream 286 (see FIG. 7) by focusing fluidstreams coming from well B, thereby flanking cells flowing from well A.The focusing fluid streams 272, 276 increase the likelihood that inputcells will enter retention chamber 270, which is placed near wherefocusing occurs. The focused stream of cells is split into two streamsadjacent the retention chamber. Each stream flows in a directionorthogonal to the focused stream and opposite to each other, asdescribed above. The trap is placed at a point of the flow stream belowwhere the stream splits, so that the velocity of flow is lower than inthe rest of the channel, therefore increasing the likelihood thatretained cells are viable. Once a sufficient number of cells arecaptured, valve 7 is closed to stop the flow of cells from well A.

[0374] System Protocols

[0375] System 250 may be used for any suitable protocols or proceduresinvolving positioned and/or retained particles. In a exemplary protocol,cells are exposed to reagents in wells D and/or E, as described below.This protocol is exemplified by successive exposure of retained cells tofirst and second reagents, such as a cell stain specific for dead/fixedcells and a cell fixative, respectively; see FIGS. 9-11.

[0376] The system is readied for perfusion as follows. First, valve 8 isclosed, so that the flow of focusing buffer from well B no longer issplit adjacent retention chamber 270. As a result, the focusing buffermoves predominantly or exclusively along main flow stream 314, which isunbranched (see FIG. 5). Next, pumps that control valve sets 354, 356are activated and run through the entire protocol. A suitable frequencyfor valve closure is about 60 Hz.

[0377] Shield buffer flow is initiated as follows. Initially, valves7-11 are in a closed position, so that only focusing buffer from well Bflows towards waste well C. Then, valve 11 is opened, so that shieldbuffer flows from F to C and focusing buffer flows from B to C.

[0378] Flow of the first reagent, in this case Trypan blue, is initiatedas follows. Valve 9 is opened, so that fluid flows through both valves 9and 11. Valves 7, 8, and 10 are maintained in their closed positions.Since the shield buffer is flowing, the first reagent is spaced from thecell retention chamber by the shield buffer. Therefore, thisconfiguration readies the system for perfusion and may be used to washthe fluidic network without exposing the cells to either of the firstand second reagents.

[0379] Perfusion of the first reagent is initiated as follows. Once thefluid lines are washed with the first reagent, the shielding buffer isturned off, and the cells are exposed rapidly to the already flowingfirst reagent. Specifically, valve 11 is closed, joining already-closedvalves 7, 8, and 10. In contrast, valve 9 remains open. In this way, theshield buffer no longer separates the flow stream of the first reagentand the cell retention chamber, allowing the first reagent to perfusethe cells.

[0380] After a suitable exposure time, the first reagent is washed outof the cell retention chamber as follows. Valve 11 is opened to restartflow of the shield buffer. In addition, valve 9 is closed to stop flowof the first reagent, joining already-closed valves 7, 8, and 10. Insome cases, valve 9 may be left open to facilitate repeated exposure ofthe cells to the first reagent over a short time interval. FIG. 9 showsabout twenty Jurkat cells 380 in retention chamber 270 after exposure toa dye, Trypan blue, that stains fixed cells and a shield buffer to washaway the dye. Debris 382 is stained, but cells 380 are unstained.

[0381] Flow of the second reagent, in this case methanol, is initiatedas follows. Valve 10 is opened, joining already-open valve 11. Valves 7,8, and 9 remain closed. This configuration is used to wash the fluidicnetwork with the second reagent without exposing the trapped cells tothis reagent.

[0382] Perfusion of the second reagent is initiated as follows. Valve 11is closed to turn off flow of the shielding buffer, joining alreadyclosed valves 7, 8, and 9. Valve 10 remains open, to expose cells 380 tothe second reagent, in this case methanol, thus fixing the cells. FIG.10 shows cells 380 being perfused with methanol. There is an opticallydetectable boundary 384 between the methanol 386 and the focusing buffer388, caused by their distinct indexes of refraction.

[0383] After a suitable exposure time, the second reagent is washed outof the cell retention chamber as follows. Valve 11 is opened to initiateflow of the shield buffer. In contrast, valve 10 is closed, to joinalready-closed valves 7, 8, and 9.

[0384] Cells 380 are then exposed for a second time to the firstreagent, followed by washing with the shield buffer, as follows. Thesequence of valve manipulations are as described above, except thatvalve 9 is left open during washing with shield buffer to show ashielded flow path of the first reagent. Now, since the cells have beenfixed and permeabilized by methanol, they stain with the dye carried inthe first reagent. FIG. 11 shows cells 380 stained blue after theirsecond exposure to Trypan blue and subsequent washing with shieldbuffer. The shielded flow path 390 of the first reagent, Trypan blue, isvisible focused between shield buffer 392 and focusing buffer 388.

[0385] The microfluidic system demonstrated here can be used for anysuitable assay, such as screening compounds against a small populationof cells, with the size of the small population be selected to bestatistically representative of cell behavior. The particles may includecells and/or beads, among others. The cells may be nonadherent and/oradherent cells, either in suspension or attached to a substrate providedby the microfluidic system. The beads similarly may be nonadherent oradherent, and may be used to carry samples, reagents, and/or cells,among others.

[0386] Embodiment 2

[0387]FIGS. 11A and 11B show a system 400 for measuring interactionbetween separated, but proximate particles. Such interaction may beprovided by diffusible materials released by a first particle (orparticle population) and received by a second, separated particle (orparticle population). These diffusible materials may includecell-secreted hormones, viral particles, cell components released bycell lysis, and/or so on. The diffusible materials may produce changesin the second particle or particle population that are related to anymeasurable particle or population characteristic, such as cell identity,gene expression, apoptosis, hormone secretion, growth, and/or the like.Alternatively, or in addition, such communication may include long, thinprocesses extending from cells, such as axons and/or dendrites.Exemplary particle characteristics are described further in SectionsVIII and XII above.

[0388] System 400 may be formed by disposing two versions of system 250in a tail-to-tail configuration. Accordingly, each individual subsystem250 may include a retention mechanism 266, an individually controlledperfusion mechanism 268 for introducing reagents to each group ofcaptured particles, and an input flow stream 274 for carrying particlesand/or buffer to the retention mechanism. However, system 400 alsoincludes communication passages 402 that provide fluidic communicationbetween each retention mechanism 266 and retention chamber 270.

[0389] Communication passages 402 may be size-selective channelsconfigured to prevent movement of retained particles, generally cells,between each subsystem 250. However, passages 402 are configured toallow movement or passage of any smaller material released from theretained particles (such as molecules, polymers, molecular complexes,and/or smaller particles, such as viruses), or of processes, such asaxons and/or dendrites, extending to, from, and/or between retainedcells. Furthermore, perfusion mechanisms 268 may be used to determinethe effect of reagents on cell-cell communication mediated by passages402.

[0390]FIG. 11B shows an alternative embodiment of paired retentionmechanisms 266, mechanism 404, that may be included in system 400.Mechanism 404 includes paired retention mechanisms 406, dimensioned totrap single particles 408. Retention mechanisms 406 are fluidicallycoupled through communication passages 402. Accordingly, communicationbetween single-cells may be analyzed using mechanism 404.

[0391] Embodiment 3

[0392]FIG. 11C shows a retention mechanism 410 that may be used insystem 250 or any other suitable microfluidic system to form apositioned, two-dimensional array of retained particles. Mechanism 410includes an array of individual traps 412 oriented to receive particlesfrom inlet channel 414. Traps 412 form a two-dimensional array ofparticle retention sites. Traps 412 may have any suitable configuration,including staggered rows, as shown here, orthogonally arranged rows andcolumns, or irregular configurations. (In some embodiments, some oftraps 412 may be positioned in alternative planes (e.g., in front ofand/or behind the plane of the drawing) to form three-dimensional arraysof retained particles.) Each trap 412 may be dimensioned to hold one orplural particles and may include size-selective channels or similarfeatures to allow fluid to flow through portions of the traps. Traps 412may be disposed within a common chamber 416 having an single or pluraloutlet channels 418 (such as chamber 270, described above, or chamber1970 of Example 10 below), within a chamber having no outlet besides aninlet channel, or within a channel, such as transverse channel 316,described above, among others.

Example 3 Microfluidic Systems for Parallel Retention and/or Treatmentof

[0393] Particles

[0394] This example describes microfluidic mechanisms and systems thatposition a plurality of particles and/or reagents at discrete transverseregions and flow paths within a channel or flow stream; see FIGS.12-13K. This positioning may allow parallel retention of distinctparticles at adjacent, but distinct, sites and/or parallel exposure ofparticles at these sites to distinct reagents.

[0395] Background

[0396] Biological analyses benefit from a capability to directly comparethe phenotypes of two or more cells or groups of cells, under similar ordistinct treatment regimens. However, in the macroscopic world, suchcells or group of cells often are treated at distinct, relatively widelyspaced sites, such as different tissue culture dishes or wells of amicrotiter plate, potentially exposing the cells to undesireddifferences in treatment conditions. Accordingly, such analyses may needto be averaged over many experiments to achieve meaningful results.Therefore, it would be desirable to have a microfluidic system thatpositions, treats, and analyzes particles or groups of particlesadjacent one another at a microscopic level, to allow more consistentand efficient side-by-side comparisons.

[0397] Description

[0398] The microfluidic systems described in this example position aplurality of particles or (particle populations) and/or reagents alongdistinct, transversely disposed flow paths or regions within a channelor flow stream. The transversely disposed flow paths may be defined byintroducing the particles and/or reagents into the channel alongdistinct laminar flow paths, by joining separate inlet channels (orinlet flow streams) carrying the particles and/or reagents. These flowpaths may abut one another or may be spaced apart by one or pluralspacer fluids, such as buffers. These spacer fluids may follow one orplural interposed flow paths formed by one or plural inlet channelsinterposed between the inlet channels that carry the particles and/orreagents.

[0399] The transversely disposed flow paths may be used to carrydistinct (or similar) particles to distinct retention sites or chamberswithin the channel. The distinct retention sites may retain distinct (orsimilar) particles for exposure to the same reagent. For example, thedistinct particles may be exposed to reagents, such as modulators and/orlabels, to compare characteristics of the particles, such as response tothe modulators, labeling characteristics, and/or so on. Thus, theposition of each retention site may be used to identify thecorresponding particle(s) retained at that position. For example, oneretention site may be used to hold a control particle(s), as areference, and another retention site may be used to hold a particle(s)of interest, allowing the control particle(s) and the particle(s) ofinterest to be compared directly. Alternatively, one retention site mayhold a bead(s) carrying a reagent, and another site may hold a cell(s)to be analyzed. In this approach, cell components released by cell lysisor secretion then may be analyzed for interaction with the reagent heldby the bead.

[0400] Alternatively, or in addition, transversely disposed flow pathsmay be used to expose similar (or distinct) particles to distinctreagents and to identify each reagent or exposed particle based onposition. Particles may be retained at positionally distinct retentionsites, either inputted from distinct reservoirs or a single reservoir.Next, the retained particles may be contacted with distinct reagentscarried to the distinct sites by transversely disposed flow paths. Thetransversely disposed flow paths may be formed by a set of inletchannels distinct from, and/or overlapping with, inlet channels thatintroduced the particles. Position of the retained particles identifieseach of the distinct reagents exposed to the particles. In someembodiments, the distinct reagents may include a compound with a knownactivity that acts as a reference, and one or more test compounds forcomparison.

[0401] The microfluidic systems of this example may allow more efficientand meaningful use of microfluidic space for comparative analysis ofparticles and/or reagents.

[0402] In certain embodiments, a junction between two inlets and anoutlet may be used to transiently expose or perfuse particles,preferably cells, with selected reagents. By alternating the inlet flowbetween plus and minus reagent flows, the downstream conditions of theoutlet will change in proportion to the rate of flow between bothinlets.

[0403] Embodiment 1

[0404]FIGS. 12 and 13 show a microfluidic system 420 (Embodiment 1) forretaining separate populations of particles, and exposing thepopulations to one or more selected reagents.

[0405] Description of Embodiment 1

[0406] System 420 is formed by multilayer soft lithography, generally asdescribed above (for system 250) in Example 2 and below in Example 13.Here, particle positioning region 422 is shown as red rectangles,input/focusing channels 424 as blue regions, and perfusion channels 426as red lines. The dimensions of each region or channel and/or the numberof channels may be selected based on particle size, reagent deliveryvolume, and/or the number of separate populations to be retained, amongothers.

[0407] System 420 differs from system 250 of Example 2 in severalaspects. First, system 420 includes more than one reservoir for holdingand introducing particles. Thus, inlets 1 and 2, shown at 428, 430,respectively, connect to particle input channels 432, 434. Second,system 420 includes three focusing channels 436, 438, 440, andcorresponding reservoirs or inlets for holding buffer (not shown). Thefocusing channels, also referred to as spacer channels, may be used toflank and separate the particle input channels. Third, system 420 hasmore than one retention chamber 442, with the chambers generallypositioned adjacent each other below confluence 444, where input flowstreams 446 join. Fourth, system 420 spaces retention chambers 442 fromwall 448, thus forming proximal and distal diverging flow streams 450and 452, respectively.

[0408] Applications of Embodiment 1

[0409] System 420 may be used as follows. Inlets 1 and 2 are loaded withdistinct suspensions of particles, such as different cell types, andinlets corresponding to focusing channels 436, 438, 440 are loaded withfocusing buffer. A pump(s) is started that drives flow of the focusingbuffer through the focusing channels. Valves that control the flow ofparticles from inlets 1 and 2 are opened. Particles enter confluence444, but are focused to spaced, intermediate, laminar flow streams 454,456, shown in FIG. 13, by flow from the focusing or spacer channels.Apertures 458, 460 of the retention chambers are aligned with particleflow streams 454) 456, respectively, to receive one or more particlesfrom the corresponding flow stream. By taking advantage of the laminarflow properties of fluids in system 420, the five streams flow togetherbut remain substantially distinct. Mixing of the fluids is limited todiffusion, which in the case of large particles, such as beads or cells,is very slow.

[0410]FIG. 13 shows the laminar flow pattern extending from confluence444 through divergence junction 462. Focusing flow streams 464, 466, 468flank and separate particle streams 454, 456, thus guiding particlescarried by these streams toward retention chambers 442. Flow streams injunction 462 may diverge above (464, 468), below (466), and/or within(470) retention chambers 442. Microchannels 472 within each retentionchamber pass fluid but retain particles.

[0411] After a sufficient number of particles have entered eachretention chamber 442, analysis of the particles may begin. Flow frominlets 1 and 2 may be terminated, and flow may be converted from adivergent pattern to a unitary flow path, by closing valve 474, asdescribed above for operation of system 250 in Example 2. Next, thetrapped particles may be perfused with buffer/reagents from perfusionchannels 426. In system 420, perfusion channel 476 discharges fluiddirectly upstream of the retention chambers. This configuration mayprovide more rapid perfusion of trapped particles with reagents thansystem 250 of Example 2 above, because the outlet end of channel 476 isvery close to the retention chambers, feeding more directly into theunitary flow path produced by the focusing buffers.

[0412] System 420 may be modified by changing various parameters. Forexample, the number of particle input-streams and/or focusing streamsmay be varied, along with the number of retention chambers, to trapadditional particle populations or individual particles. Thus, three ormore particle input-streams may be used to trap three or more types ofparticles in three or more retention chambers. These three or moreretention chambers may be disposed in any suitable arrangement,including linear and staggered (e.g., triangular configurations). Insome embodiments, the size of the retention chambers may be varied, forexample, so that only one or a very small number of particles aretrapped in each chamber (see embodiment 2 of this example, and Examples4-7, 11, and 12 below). Furthermore, as described below, focusingstreams and spacer channels may be eliminated in some cases withoutsubstantial cross-contamination of particles between particle streamsand retention sites.

[0413] Embodiments 2 and 3

[0414] FIGS. 13A-C shows two alternative embodiments of system 420,systems 480 (Embodiment 2) and 480′ (Embodiment 3), for retaining andtreating particles at separate, but adjacent sites. Similar to system420 described above, system 480 or 480′ may be used to selectively inputand retain one or plural particles at each of plural retention sitespositioned at discrete positions transverse to a flow direction within achannel. However, system 480 or 480′ also may be used to separatelycontact retained particles with distinct reagents at distinct retentionsites.

[0415] Description of Embodiments 2 and 3

[0416] System 480 includes an input mechanism 482, a focusing ortransverse positioning mechanism 484, a retention mechanism 486, anoutput mechanism 488, a plurality of individually controllable anddistinct treatment mechanisms 490, 492, and a release mechanism 494; seeFIGS. 13A and 13B.

[0417] Input mechanism 482 includes particle input channels 496, 498 andfocusing or spacer channels 1762, 1764, 1766, similar to those describedabove for system 420. Particles, such as cells, may be inputted frominput reservoirs “Cell 1” and “Cell 2” along particle inlet channels496, 498, to positioning channel 1768. Input mechanism 482 also mayintroduce focusing or spacer fluid, preferably buffer, from bufferreservoirs 1770, 1772, 1774 (“Buffer 1,” “Buffer 2,” and “Buffer 3,”respectively) along spacer channels 1762, 1764, 1766, respectively, topositioning channel 1768.

[0418] Transverse positioning mechanism 484 may be determined by inletchannels. More specifically, the relative spatial configuration in whichthe inlet channels 496, 498, 1762-1766 join positioning channel 1768,along with relative sizes of, and/or flow rates from, these inletchannels, provides transverse positioning mechanism 482. Positioningmechanism 484 places each individual flow stream from each inlet channelin a laminar flow path based on this spatial configuration. Accordingly,particles from reservoirs Cell 1 and Cell 2 are spaced from each othercentrally in positioning channel 1768 by buffer from inlet channel 1764and laterally from each channel wall by buffer from inlet channels 1762,1766, as described above for system 420.

[0419] Retention mechanism 486 includes a plurality of single-particleretention sites, here referred to as “Trap A” and Trap B” (see FIG.13B). Trap A and Trap B each are positioned to retain a particleintroduced by one of the two particle reservoirs, Cell 1 and Cell 2, andcarried at correspondingly distinct, transverse positions alongpositioning channel 1768; see FIG. 13B. Accordingly, Trap A ispositioned to retain a particle introduced from Cell 1, and Trap B fromCell 2. Particles not retained may be carried past retention mechanism486 to output mechanism 488, along central outlet (waste) channel 1776or flanking outlet (waste) channels 1778.

[0420] Treatment mechanisms 490, 492 provide exposure of retainedparticles to distinct reagents, indicated as Reagents 1-4; see FIG. 13B.A particle retained in Trap A may be exposed to Reagent 1 and/or 2(controlled by valves V2 and V3), and a particle retained in Trap B maybe exposed to Reagent 3 and/or 4 (controlled by valves V6 and V7). Thesereagents may be stored and delivered (sequentially and/orsimultaneously, in any desired proportion and for any desired time)using any suitable mechanism, such as those described above in Example 2and below in Example 8. Reagents from each treatment mechanism may beseparately addressed to a corresponding retention site, by transversepositioning of reagent flow streams entering positioning channel 1768.Reagents flow toward central outlet channel 1776, but occupy a discreteportion of the entire flow stream within positioning channel 1768 andtransverse channel 1780 due to laminar flow. Accordingly, reagents fromtreatment mechanism 490 may be restricted to the left side ofpositioning channel 1768 in FIG. 13B (and thus Trap A), whereas reagentsfrom treatment mechanism 492 may be restricted to the right half of thechannel (and thus Trap B). Optionally, spacer buffer from centralreservoir 1772, Buffer 2, may flow between reagents delivered by thetreatment mechanisms, reducing the probability of any reagent crossingover, and thus contaminating, the noncorresponding retention site.

[0421] Release mechanism 494 enables release of retained particles.After release, the released particles may be analyzed further and/orcollected, and/or the retention sites may accept a new set of particlesfor another round of treatment and analysis. Release mechanism 494, maybe operated by valve V4, to produce a localized reverse or dislodgingflow that propels the retained particles out of the retention sites.Release mechanism 494 is similar to the release mechanism describedbelow in Example 7. However, in contrast to the release mechanismdescribed below, retention sites in the present example are spaced fromreverse flow channels 1782.

[0422]FIG. 13C shows selected portions of a modified version of system480, system 480′. System 480′ is distinct from system 480 in at leasttwo aspects. First, retention mechanism 1784 includes retention chambers1786, 1788 that are larger than the retention sites of system 480, andthus are capable of holding plural particles. Second, treatmentmechanisms 1790, 1792 include reagent inlet channels 1794, 1796 thatintroduce reagents into transverse channel 1798, rather than positioningchannel 1800. This altered position of the reagent inlet channels movesthe reagents farther from retained particles, but may facilitate washingout reagents toward outlet channels 1802 after exposure. However, duringtreatment, reagents from inlet channels 1794, 1796 are still positionedtransversely relative to the general direction of fluid flow towardcentral outlet channel 1804. Accordingly, reagent inlet channels maydeliver reagents at any suitable sites that allow laminar flow-basedlocalization of reagents.

[0423] Systems 480 and 480′ may be modified in any suitable aspect. Forexample, a single population of particles, such as from a single inputreservoir, may be retained at plural distinct retention sites, such asTrap A and Trap B, and then the sites separately exposed to distinctreagents introduced by distinct treatment mechanisms. Alternatively, orin addition, inlet channels provided by treatment mechanisms andparticle input mechanisms may overlap or converge upstream of a commonpositioning channel, such as positioning channel 1768 or 1800.

[0424] Applications of Embodiment 2

[0425] Exemplary operation of system 480 is described below using cells.System 480 may be readied for operation by loading the input reservoirswith cells and buffers and equilibrating channels with the buffers, asdescribed in other examples.

[0426] Trap A and Trap B may be loaded as follows. Valves V1, V4, and V5are opened, and valves V2, V3, V6, and V7 are closed. Five flow streamscoming from each of the five reservoirs meet before Trap A and Trap B inpositioning channel 1768. The cells from reservoirs Cell 1 and Cell 2are directed to their respective Traps A and B. Fluid and unretainedcells flow past retention sites along divergent flow paths toward aplurality of outlet channels 1776, 1778.

[0427] Once a cell (or cells) is retained in each retention site, valveV4 is left open, and valves V1 and V5 are closed. Closing valve V1blocks input of additional cells, and stops flow from lateral bufferreservoirs 1770, 1774. Closing valve V5 stops divergent flow, so thatbuffer (from central buffer reservoir 1772 (Buffer 2)) flows to centraloutlet channel 1776 along a unitary path.

[0428] Distinct reagents may be delivered to the retained cells asfollows. Valve V4 is left open, and all other valves remain closed. Bothpumps are running. Valve V2 and/or valve V3 may be opened to addressReagent 1 and/or 2 to Trap A. Valve V6 and/or valve V7 may opened toaddress Reagent 3 and/or 4 to Trap B. Valves may be partially opened asdescribed in Example 8 to provide a desired mixture of reagents. Bufferfrom reservoir 1772 flows past Traps A and B to outlet channel 1776 andmay be used as a barrier between the streams of reagents addressed toTraps A and B. At any suitable time, valve V5 may be closed to releasethe retained cells.

[0429] Exemplary Results with Chips Produced According to Embodiment 2

[0430] System 480 was tested as described below. Microfluidic chips werefabricated according to system 480 of FIG. 13A and used for analysis offlow patterns and particle treatment efficacy with colored and/orfluorescent dyes.

[0431] FIGS. 13D-F show dye patterns formed by colored dyes introducedusing each treatment mechanism and a flowing spacer buffer to separatereagents. In each figure, Trap A holds a 10 μm bead, and Trap B two 6 μmbeads. FIG. 13D shows a dye pattern formed by green dye delivered fromeach treatment mechanism and an orange dye-labeled spacer bufferdelivered by reservoir 1772. The orange spacer buffer separate the twogreen dyes, and each green dye flows from its corresponding inletchannel 1806, 1808 to outlet channel 1776. Some green dye also travelsslowly along transverse channel 1780. FIG. 13E shows a dye patternformed by red dye delivered from treatment mechanism 490, green dye frommechanism 492, and orange dye from buffer reservoir 1772. FIG. 13F showsa dye pattern formed by red dye delivered from treatment mechanism 490,yellow dye from mechanism 492, and orange dye from buffer reservoir1772.

[0432] FIGS. 13G-13J show an analysis of treatment efficacy of singleJurkat cells captured in each of Traps A and B. FIG. 13G shows the tworetained cells 1810, 1812 prior to treatment. FIG. 13H shows exposure ofeach cell to Trypan blue dye delivered by distinct treatment mechanisms.The spacer buffer forms an uncolored column of fluid 1814 between thetwo blue regions surrounding Traps A and B. Membranes of both cells areintact so neither stains efficiently with the dye. FIG. 131 showsexposure of cell 1810 in Trap A to methanol, to fix the cell, while cell1812 in Trap B is addressed with buffer. FIG. 13J shows the two cellsbeing exposed to the blue dye after fixation of cell 1810. Cell 1810 canno longer exclude the blue dye and is stained blue. Cell 1812 has notbeen in contact with methanol and is not stained.

[0433]FIG. 13K demonstrates that spacer buffers may not be required toprevent cross contamination of particles and/or reagents during particleloading and/or exposure to reagents. Each trap has been loaded with afluorescent bead 1816, 1818. Bead 1816 is addressed with a fluorescentdye, fluorescein, and bead 1818 with Trypan blue, using treatmentmechanisms 490, 492, respectively. No spacer buffer stream separates thetwo reagent streams, but the reagents do not substantially cross overand contaminate the other trap. It should be noted that the time fordiffusion of reagents (or particles) transverse to their laminar flowstreams is limited by the relatively short time that the laminar flowstreams are in contact before passing Traps A and B. Accordingly,analyses may be conducted with or without spacer streams, with spacerstreams being used to lower the probability of cross-contamination.

[0434] Embodiment 4

[0435]FIG. 13L shows a portion of microfluidic system 1820 that may beused to separately address particles and/or reagents to sets of particletraps. System 1820 includes a plurality of serially arrayed sets 1822,1824 of particle traps 1826. Each set 1822, 1824 is disposed to adiscrete transverse position with a fluid flow stream, in this casedefined by a channel 1828. Accordingly, laminar flow streams carryingparticles (1830, 1832) or reagents (1834, 1836) may be segregated todiscrete transverse regions of channel 1828, so that each set 1822, 1824is individually addressed. In alternative embodiments, traps 1826 aredisposed in a transverse channel, such as channel 1798 or a chamber,such as a cell chamber with size-selective channel around its perimeter.

Example 4 Microfluidic System for Multiplexed Analysis of Particles inan Array

[0436] This example describes a microfluidic system that loads particlesin a serially distributed set of particle retention sites, andseparately addresses reagents to each of these sites in parallel; seeFIGS. 14-16.

[0437] Background

[0438] Cell analyses often involve the use of arrays of cells or cellpopulations. These arrays may be formed in microtiter plates, so thatindividual wells within the array can be treated distinctly, forexample, with distinct test compounds. During or after treatment, themicroplate arrays are analyzed in multiplex to measure properties ofcells within each individual well. However, such arrays are difficult toform reproducibly with microtiter plates when single cells or a smallgroup of cells are placed in each well. Even if formed in microtiterplates, rapidly treating the cells in such microtiter plates, andmeasuring short-term consequences of such treatments, poses substantialtechnical hurdles. Therefore, a microfluidic system is needed that formsmore reproducible arrays of individual cells or small groups of cells atdistinct positions, and that allows separate, rapid treatment andanalysis of the cells at the distinct positions.

[0439] Description

[0440] This example describes a microfluidic system that serially trapssmall sets of particles at preselected positions within the system,allowing treatment of the trapped particles in parallel with desiredreagents. Due to serial trapping of input particles, a single loading ofparticles into one inlet may be used to supply particles to an entirearray of traps. Thus, this design may be used to integrate a largenumber of traps into a single system. This microfluidic system alsoreduces the number of control lines required, as single control linesregulate sets of fluidic channels, such as perfusion channels, thatindividually interface with each of the traps. Accordingly, singlecontrol lines provide parallel control for fluidic delivery to, oroutput from, each of the traps. Such parallel control allows similarparticles that are retained by each trap to be individually treated withdistinct reagents. Furthermore, such parallel control allows all trapsto be fluidically connected during particle loading, but thenfluidically isolated during particle treatment and measurement. Thisarrangement of the traps enables the fabrication of larger microfluidicsystems that may be suitable for use in high-throughput drug discovery.For example, system 510 has a footprint of 2 by 4 cm. By increasing thisdensity somewhat and increasing the number of traps over twenty-fold, atleast 128 traps may be disposed on a single substrate of 8 by 12 cm,allowing each of the 128 traps to be addressed by two distinct reagents,with a total of 256 reagents per substrate.

[0441]FIG. 14 shows a microfluidic system 510 for forming and analyzingan array of particles. System 510 may be formed by any suitabletechnique, such as multilayer soft lithography, to include at least twodistinct layers: (1) a microfluidic network layer 512, shown in blue andorange, and (2) a control layer 514, shown in pink. Channels havingdistinct widths and/or cross-sectional shapes may be formed within eachlayer using molds fabricated, for example, as described in Example 17.

[0442] Microfluidic layer 512 includes two orthogonally directednetworks. Particle loading network 516 is used to input and positionparticles, so that the particles are retained at a linear array ofparticle traps 518. Particle treatment system 520 is an array ofparallel, individual perfusion networks 522 that intersect loadingnetwork 516 at individual particle traps 518.

[0443] Particle loading network 516 includes an inlet 524, an outlet526, and a loading channel 528 extending there between. Inlet well 524,labeled C, is a reservoir that receives and holds a particle suspensionto be introduced into network 516. Outlet well 526, labeled W, is awaste reservoir that receives and holds fluid and unretained particlesthat have traveled through network 516. Loading channel 528 carriesparticles between inlet well 524 and outlet well 526 to each of aplurality of particle traps 518 disposed along channel 528. Fluid isactively transported along network 516 by a three-valve pump 530,labeled “pump 1,” which is positioned near the terminus of network 516to pull fluid through the network. Positioning the pump after the trapsdelays potential damage to fragile particles, for example, due tocompression under closing valves, until particles have passed allparticle traps 518.

[0444] Each perfusion network 522 directs fluid between perfusion inlets532, traps 518, and treatment outlets 534. Perfusion inlets 532 are oftwo main types: buffer inlet-wells 536, labeled “B,” and reagentinlet-wells 538, labeled “Ry+.” The buffer inlet-wells hold a buffer orother washing or maintenance liquid, such as water or a solvent. Basedon their positions within particle treatment system 520, the bufferinlet-wells are either a terminal inlet-well 540 or an intermediateinlet-well 542. Terminal inlet-wells 540 feed fluid to only one trap,whereas intermediate inlet-wells 542 are shared between two adjacenttraps. Based on whether they are intermediate or terminal inlet-wells,buffer inlet-wells feed a main stream and/or a shielding stream. Thecontrol and function of these two streams are described further below.The reagent inlet-wells hold one of two (or more) reagents (or reagentmixtures) that may be precisely exposed to an individual trap. Reagentinlet-wells are labeled “R_(xy),” with “x” referring to trap assignmentrelative to the array of traps 518, and “y” referring to one of the tworeagents that can be directed to a given trap. For example, reagentinlet-well R₁₂ feeds the first of the plurality of traps (closest inletC) with the second of two reagent choices for that trap. Fluid thatpasses each trap 518 may be directed to a corresponding treatmentoutlet-well 534 or waste well, labeled here as W1-W6. For example,reagents from reagent inlets R₄₁ and R₄₂ flow past and/or through trapnumber 4 and are collected in waste well W_(x), where x=4.

[0445] Control layer 514 regulates fluid flow from perfusion inlet-wells532 with a limited number of control lines that act on many fluidchannels 544 in parallel; see FIGS. 14 and 15. A three-valve pump 546,“pump 2,” acts simultaneously on all inlet channels 544 that extend fromperfusion inlet-wells 532, to actively drive fluid from theseinlet-wells to and past traps 518, and on to waste outlet-wells 534.Opening or closing each of four perfusion valves, V1-V4, determineswhether fluid actually flows through each of the specific types of inletchannels 544 within the perfusion system. Valve V1 regulates controlline 548, which includes a plurality of individual valves positionedover each of a corresponding plurality of focusing channels 550 includedamong inlet channels 544. Similarly, valve V2 regulates control line552, which includes valves that control each of a plurality offirst-reagent channels 554, valve V3 regulates line 556, which controlseach of a corresponding plurality of second-reagent channels 558, andvalve V4 regulates line 560, which controls each of a correspondingplurality of shield channels 562. Thus, opening or closing each ofvalves VIV4 provides unified, parallel control over flow of individualinlets to each of the plurality of traps.

[0446]FIG. 15 shows a portion of system 510, including traps 2, 3, and4, to illustrate in more detail the design and rationale for theswitching valves. Insulation valves 564 function in the control layer tomediate switching between particle loading network 516 and particletreatment system 520. Insulation valve V5 controls a set of valves thatblock flow along loading channel 528 at a position downstream ofparticle inlet 524 (inlet C) and of the traps. Thus, activation of valveV5 fluidically isolates each trap and converts system 510 from aparticle-loading configuration to a perfusion configuration. Incontrast, insulation valve V6 controls a set of valves blocking flow toeach individual treatment outlet 534, preventing diversion of particlesto treatment outlets during particle loading, when valve V6 is closed.Therefore, valves V5 and V6 are primary determinants of parallel versusserial use of system 510.

[0447]FIGS. 15 and 16 show details of the loading mechanism. Loadingchannel 528 forms a divided flow path 564 at each trap 518. Thus,particle stream 566 diverges directly upstream of each trap 518, at aT-junction 568, following divided flow path 564, and then converging toform reunited particle stream 566. At each T-junction 568, a subset ofparticles do not follow divided flow path 564, but flow instead directlyinto trap 518. Accordingly, each trap is loaded using a divergent-flowmechanism, as described above in Example 2, but, in system 510, withoutthe use of focusing-buffer streams during particle loading to focusparticle flow within channel 528. In this example, trap 518 includes aretention chamber similar to retention chamber 270 of FIGS. 5-8 inExample 2. However, any suitable traps may be used, such assingle-particle traps described below in Examples 4-7, 11, and 12.

[0448] The subsequent perfusion of trapped particles uses shielding andperfusion mechanisms analogous to those of Example 2. Buffer flow fromeach buffer inlet 536 flows along focusing channels 550, into loadingchannel 528, and past trap 518 in a unitary flow path 572, shown in FIG.16 as a dashed path, analogous to focusing buffer stream 314 of FIG. 5.Unitary flow path 572 may perform a variety of functions, such asbathing trapped particles during treatment, providing a retaining forceon trapped particles during perfusion, and focusing inflowing reagentsand shield buffer, in their laminar flow streams, toward the trappedparticles. Similarly, combined first and second reagent channel 554/558and shield channel 562 determine precise exposure to first and secondreagents, as described above in Example 2.

[0449] Applications

[0450] An exemplary use of system 510 to load particles and expose theparticles to different reagents is described below. System 510 is formedand readied for use as described elsewhere in this Detailed Description.

[0451] Loading particles into each of traps 518 may be conducted asfollows. Valves 1-4 and 6 are closed, and valve 5 is open. Pump 1 isrunning, and pump 2 is not. The buffer inlet-wells B, shown at 536, areloaded with buffer, each of inlet-wells R_(xy) is loaded with a reagent,and inlet-well C is loaded with a cell suspension. After making surethat the waste inlet-wells 526 are empty, pump 1 is allowed to pull theparticles to the traps.

[0452] Conversion from a loading to a perfusion configuration may becarried out as follows. Once each of the traps has its desired occupancyand/or is full, pump 1 is stopped and valve V5 is closed. Each trap isnow isolated. Next, Valve V6 is opened to allow fluidic access to wasteoutlet-wells 534. Then, valve V1 is opened to permit flow of buffer fromeach inlet-well 536.

[0453] Trapped particles are perfused with each of the first and secondreagents as follows. Pump 2 is started, running at a frequency of about60 Hz. This pump is running throughout the following treatments. Pumpingaction of pump 2 drives buffer through focusing channels 550, alongunitary flow path 572 past each trap 518, toward waste outlet-wells 534.Prior to perfusion, valves V2, V3 and V4 are closed, so that only nofluid flows from along shield channel 562 or reagent channels 554, 558.Flow of the first reagent and the shield buffer is initiated by openingvalves V2 and V4, while valve V3 remains closed. This valveconfiguration is used to wash the fluidic network without exposing thetrapped particles to the first reagents, because the shield bufferdirects the first reagent stream to a spaced flow path separated fromthe trapped particles. Once the fluid lines are washed with each of thefirst reagents, valve V4 is closed to stop from of the shield buffer,allowing each of the first reagents to contact trapped particles. Aftera desired duration of exposure to each first reagent, valve V2 isclosed, allowing the shield buffer to wash away reagent one, and rapidlyterminating exposure. Trapped particles may be exposed to each secondreagent in parallel by following a comparable series of steps, butopening and then closing valve V3 instead of V2. In alternativeperfusion strategies, particles may be exposed to both the first andsecond reagents simultaneously, by opening both valves V2 and V3together. Furthermore, particles may be exposed to any desired ratio offirst and second reagents by partially closing valves V2 and/or V3, asdescribed below in Example 7.

Example 5 Microfluidic Device for Forming and Analyzing a Particle ArrayUsing a “Cell Comb”

[0454] This example describes a microfluidic device for forming andanalyzing arrays of small number of particles, such as cells; see FIGS.17-20.

[0455] Background

[0456] In many applications, it is necessary to form an array ofcell-analysis chambers, with each chamber containing the same number ofcells. These chambers allow multiple experiments, such as drug screens,to be conducted in parallel, in a consistent and comparable fashion.Currently, standard analyses use wells of microtiter plates as cellchambers, distributing an equal volume of a cell suspension to each ofthe wells. The size of these chambers and thus the number of cellsanalyzed has been decreasing in response to efforts to reduce the use ofspace, reagents, and cells in these analyses. Unfortunately, resultsfrom these analyses become increasingly variable as the average numberof cells per well decreases. For example, with 96-well microtiterplates, there generally are about 3000 to 5000 cells at the bottom of awell; with 384-well plates, this number drops to about 1000 cells; and,as researchers push for smaller and smaller assay volumes, such as with1536-well plates, this number drops further to only about 250 cells.These small average numbers of cells may lead to variations in theactual number of cells among wells of as high as 20%. Such variationslead to huge errors in the detected reaction signals. Accordingly, witheven fewer cells per well, for example, with single cell assays or whencells of interest are in limited supply, microtiter plates do notprovide an adequate cell-analysis chamber unless cells are counted toplace an equal number per well. Even then, microtiter plates aredeficient for performing rapid experimental manipulations. For example,early responses to treatment with a drug are difficult to measure withmicrotiter plates, because adding and mixing steps cannot be performedvery rapidly. Therefore, many cell-analyses would benefit from systemsfor efficiently loading, rapidly treating, and analyzing small numbersof cells.

[0457] Description

[0458]FIG. 17 shows a microfluidic device 610 for forming an array ofsingle particles or small groups of particles. Device 610 includes aninput channel 612, a waste channel 614, and an array of filter channels616 extending between the input and waste channels. Device 610 alsoincludes a fixed-volume particle chamber 618 formed in each filterchannel 616, and a set of valves for sample handling (see below). Device610 may be referred to as a “cell comb” because the path for cell(particle) flow takes the shape of a comb, with chambers 614representing the teeth of the comb.

[0459] The components of a cell comb each have a distinct function.Input channel 612 carries input particles, such as a particle 620, toeach filter channel 616. A filter 622 is disposed within, or adjoining,each filter channel. Filter 622 allows fluid to pass into waste channel614, but retains particles 620 in a portion of filter channel 616 thatcorresponds to chamber 618.

[0460] Filter 622 may take various forms, provided as a component(s)separate from the walls of filter channel 616 and/or integral to thesewalls. For example, filter 622 may be formed by a porous membrane thatis specific for each chamber 618 or that is shared by two or more or allchambers 618. Alternatively, filter 622 may be formed by smaller, “leak”channels within filter channel 616, or by posts, obstacles, orprotrusions that extend into a portion of filter channel 616, or thatare disposed adjoining or adjacent an end of the filter channel. Thediameter of the smaller channels, or the spacing of the posts/obstacles,determines the size of particle retained in chamber 618. Thus, as longas the diameters of these smaller channels, or the maximum spacingbetween these posts/obstacles, are sufficiently less than the diameterof a particle to be retained, the particle will be confined to chamber618 while fluid will pass readily into waste channel 614. In addition,the passage of fluid through the filter provides a retaining force toreduce or prevent backflow of particles into input channel 612.

[0461] The capacity and retention ability of each chamber 618 is definedat least in part by filter channel 616 and filter 622. The diameter andlength of filter channel 616, coupled with the position of filter 622relative to filter channel 616, define the capacity of chamber 618.Accordingly, chamber 618 may be dimensioned to receive a fixed number ofinput particles 620, such as a single particle. Such input particles mayhave a common size, such as cells from a homogeneous cell population, orthey may have a range of sizes, such as cells from blood. In someembodiments, the diameter of filter channel 616 allows size-selectiveretention of a single particle. For example, the diameter may be largeenough to receive certain particles in a heterogeneous particlepopulation, such as red blood cells, but small enough to exclude others,such as white blood cells. Filter 622 also acts size selectively, asdescribed above, so in combination with chamber 618, individual filterchannels 616 may be designed to retain a single cell within a definedsize range. Alternatively, individual filter channels may be designed toretain a group of two or more cells, with each cell having a minimumsize that is retained by filter 622.

[0462] Pressure differences within device 610 create positioning andretaining forces for particles 620. Flow between input channel 612 andwaste channel 614 creates a positive pressure difference between theinput channel and the waste channel across filter channel 616. As aresult, particles are carried into chambers 618 by fluid and fill eachof the chambers very rapidly. After the particles have filled some orall of chamber 618, a set of valves may be used to isolate each chamber618 (see below). In particular, the closure of such valves may transformeach cell chamber into an isolated reaction chamber, with a fixed numberof particles for analysis.

[0463] FIGS. 18-20 show valves, additional filters, and analysis sitesthat may be used with, or added to, device 610 for manipulating thecontents of individual chambers 618.

[0464]FIG. 18 shows a device 630 that is similar to device 610, but thatincludes a separate analysis site 632 opposing each chamber 618. A sitevalve 634 controls access to analysis site 632, and a pair of inputvalves 636 isolates each chamber 618 along input channel 612. The leftpanel of FIG. 18 shows a loading configuration for each of valves 634,636. Here, site valve 634 is closed (indicated by an “X”) to preventinput particles 620 from entering analysis site prematurely, and inputvalves 636 are open to allow particles to access each chamber 618. Theright panel of FIG. 18 shows repositioning of retained particle 620 toanalysis site 632. Here, site valve 634 is open, but input valves 636are closed. Particle 620 is displaced from chamber 618, by fluid flowingin reverse across filter channel 616 from waste channel 614, rather thaninput channel 612. Since input valves 636 are closed, fluid and particle620 flow orthogonally to input channel 612, into analysis site 632.After particle 620 is delivered to analysis site 632, site valve 634 isclosed to isolate the particle fluidically from other particles. Inother embodiments, additional fluidic lines may be used to deliverreagents to analysis site 632, or analysis site 632 may be a blindchannel that is preloaded with such reagents.

[0465]FIG. 19 shows a device 650 that is similar to device 630 of FIG.18, but that includes switchable filters 652. Switchable filters 652 maybe switched between a closed, filtering position, shown on the left, andan open, nonfiltering position, shown on the right. After particleloading, switchable filters 652 are opened to direct particle 620 to ananalysis site. Such a switchable-filter design allows unidirectionalflow across filter channel 616 to both retain and release particle 620.Accordingly, fluid flow from input channel carries out each bothretention and release, using particle-laden fluid during retention, andparticle-free fluid during release. Waste valves 654 are closed beforeswitchable filter 652 is opened to direct particle 620 to analysis site656. Switchable/regulatable filters may be formed by size-selectivechannels that are formed on valve membranes. With this arrangement,deflection of the valve membranes may move the size-selective channelsin or out of filtering position by pressure exerted through a controllayer. Alternatively, or in addition, size-selective channels may beadjacent to, or flanking, valve membranes, as described below in Example26.

[0466]FIG. 20 shows another device 660 with a switchable filter 652. Indevice 660, waste channel 614 includes a series of waste filters 662that function in place of waste valves 654 in device 650. Waste filters662 play a dual role in allowing waste to flow down waste channel 664,while directing particle 620 toward analysis site 666. The passages ofanalysis sites 666 may serve as waste channels.

[0467] Applications

[0468] Cell combs, described in this example, may be useful in a varietyof applications. For example, cell combs may be useful in drugdiscovery, serving as replacements for microtiter plates in cell assaysto provide tighter control of the cell numbers. With current technology,the fabrication of each cell chamber in a cell comb device can becarried out with precision. Therefore, cell assays may be performed withan array of cells formed using this device, with reduced signalvariation from chamber to chamber, even with single-cell assays. Cellcombs may, more generally, be used with a variety of micron-sizedparticles, in addition to cells, such as fluorescently or enzymaticallycoated beads. This device also can operate in gas phase, as long as thesize of the particles of interest is larger than the pore size of thefilter units. Cell combs also can be cascaded so that objects ofdifferent sizes are filtered out at different stages.

Example 6 Particle-Retention Mechanisms

[0469] This example describes mechanisms for retaining particles, usingparticle traps that are spaced from their corresponding substrates; seeFIGS. 21-23.

[0470] Background

[0471] One goal of microfluidic systems is the capability of retainingparticles at preselected positions for subsequent treatment andanalysis. Traps that perform such retention functions may performoptimally if they have minimal effects on fluid flow; otherwise, flowpatterns around the traps may be disrupted, slowing or reducing particleand reagent entry into the traps. Examples 1 and 2 above describe trapsthat may be used to retain single particles or groups of particles.However, these traps have limited flow through the traps themselves. Forexample, trap 180 of Example 1 includes blocks P and Q that reduce orprevent cross-flow on either side of a single retained particle.Similarly, retention chamber 270 of Example 2 includes relatively narrowmicrochannels 300 that may restrict fluid flow substantially. Thus,there is a need for an alternative trap that may be positioned closer toparticle input flow streams without disrupting flow patterns, whileallowing quicker and more efficient access by reagent and washing flowstreams.

[0472] Description

[0473] This example describes retention mechanisms having improved fluidflow properties. These mechanisms are positioned downstream of aparticle flow stream, near the point at which the particle flow-streamdiverges at a T-junction. These mechanisms have been dimensioned to trapa single particle; however, they alternatively may be dimensioned totrap two or more particles. The microfluidic system with respect towhich each retention mechanism is illustrated, particularly positioningmechanism 264 and perfusion mechanism 268, is described above in Example2. This earlier example describes suitable fluid flow paths, and theoperation of the positioning and perfusion mechanisms. However, theretention mechanisms presented in this example may be combined with anyother suitable microfluidic mechanisms for particle analysis.

[0474] Embodiment 1

[0475]FIG. 21 shows a microfluidic system 710 for positioning,retaining, and/or perfusing a single particle, in accordance withaspects of the invention. Portions of system 710 that are molded fromdistinct photoresist layers are shown as distinct colors, as describedabove (see introductory section of Examples). Retention mechanism 712includes a trap 713, shown in turquoise, positioned centrally inT-junction 714, in a spaced relation from distal wall 716. Here, view718, on the top right, is a schematic representation of trap 713, withpoints of sectional view indicated; view 720, on the middle right, is ahorizontal sectional view near the top of retention mechanism 712; andview 722, on the bottom right, is a vertical sectional view nearer theside of retention mechanism 712. Trap 713 extends downward from roof 724as a U-shaped block 726. This block includes a recess 728 that acts as aretention site for a single particle. The block extends toward substrate730, in this case formed of glass, but remains in a spaced relation, inthis case about 5 μm apart from the substrate, to form a flow channel732 that extends under all of block 726. Thus block 726 forms astalactite-based trap with a potential flow stream below its entirebottom surface 734.

[0476] Embodiment 2

[0477]FIG. 22 shows another microfluidic system 740 for positioning,retaining, and/or perfusing a single particle, in accordance withaspects of the invention. View 742 shows a color-coded schematic of asystem 740, whereas view 744 shows a photograph of an actualmicrofluidic system formed according to view 742, but flippedhorizontally. System 740 includes a trap 746 positioned centrally atT-junction 714. Trap 746 is spaced from distal wall 716, disposing anyretained particle quite close to perfusion channel 748 for very rapidexposure to reagents (see Example 2 for a more complete description ofthe perfusion mechanism). Trap 746 includes a retention site 750 forholding a particle, flanked by trap channels 752, shown in turquoise,that extend to the edges of trap 746. Thus, fluid can enter retentionsite 750 and flow laterally out trap channels. View 754 shows thestructure of trap 746 schematically. Trap 746 includes three rectangularcolumns 756 that extend down to substrate 730, bridged by channelforming portion 758, shown in dotted outline in view 754, which extendsdown to 5 μm from substrate 730. Cross-sectional views 762, 764, 766show the structure of trap 746 in more detail.

[0478] Embodiment 3

[0479]FIG. 23 shows yet another microfluidic system 790 for positioning,retaining, and/or perfusing a single particle, in accordance withaspects of the invention. System 790 includes a particle retentionmechanism, trap 792, that abuts distal wall 716, in alignment withparticle stream 794 focused down input channel 796. Trap 792 includes aretention site 798, which is twenty μm in height, and flanked byretention blocks 800 that are spaced from substrate 730 by about 5 μm.View 802 shows a line representation of trap 792, but includes a portion804 of microfluidic system outside of distal wall 716. Sectional views806, 808 show how retention blocks 800 extend outward and downward fromdistal wall 716 and channel roof 810, but form a trap channel 812 thatextends under entire bottom surface 814 of the trap. Thus, trap 792 isstructured as a stalactite.

[0480] Views 816, 818 are two photographs taken of trap 792 at differentdepths of focus. In view 816, the focal plane is near the substratesurface, showing sharp lines at corners 820, where the microfluidiclayer 822 contacts substrate 730. The bottom perimeter 824 of blocks 800is blurry because bottom surface 814 is raised above substrate 730 (seealso views 806, 808). In view 818, the focal plane is slightly higher,raised about 5 μm, placing bottom perimeter 824 in focus. Now, corners818 are out of focus.

Example 7 Mechanisms for Reusable Microfluidic Systems

[0481] This example describes mechanisms that promote reuse ofmicrofluidic systems, including mechanisms for release, collection,and/or resuspension of particles; see FIGS. 24-28.

[0482] Background

[0483] Microfluidic systems often are designed for single use. Suchsingle-use systems may be used to retain and analyze a single cell ormultiple cells, but they then are not or cannot be used again becausethe cell or cells interfere with analysis of newly introduced cells.Thus, these single-use systems then are discarded, and additionalsingle-use systems must be initialized for additional analysis. Thisapproach is not an efficient use of the single-use systems. Moreover,this approach wastes macroscopic volumes of cells and reagents, and istime consuming for initialization. Thus, there is a need for a reusablemicrofluidic system that releases retained particles after theiranalysis, freeing the system (or cells) for additional analysis.

[0484] Description

[0485] This example describes microfluidic mechanisms that enableformation of reusable microfluidic systems. These microfluidicmechanisms include (1) a particle release mechanism, (2) a particlecollection mechanism, and (3) a particle suspension mechanism. Theparticle release mechanism removes a particle(s) from a trap, generallyafter treatment and/or analysis in the trap. The release mechanism mayprovide a force that propels particles out of the trap at any selectedtime. The particle collection mechanism may be used to collect particlesdischarged by the release mechanism. Collected particles may becultured, measured, treated, and/or discarded. The particle suspensionmechanism reduces particle settling in an inlet well, so that a singleloading of particles into the inlet well produces a relatively constantparticle flow from the inlet well over time. These three mechanismsalone, or in any suitable combination, may enable more efficient andeconomical use of microfluidic systems for particle analysis.

[0486] Embodiment 1

[0487]FIG. 24 shows a microfluidic system 850 having a particle releasemechanism 852 and a particle collection mechanism 854, in accordancewith aspects of the invention. The general design of system 850 is asdescribed in Example 2, and elsewhere in this Detailed Description,including a particle focusing mechanism 856, a particle retentionmechanism or trap 858, and a perfusion mechanism 860. These particlefocusing and perfusion mechanisms are at least substantially equivalentto positioning and perfusion mechanisms 264, 268, respectively, shown inFIG. 5 of Example 2. System 850 may be formed as described elsewhere inthis Detailed Description. The meaning of each colored region of system850 also has been described above, and therefore will not be repeatedhere.

[0488]FIG. 25 shows trap 858 in more detail. Trap 858 may be dimensionedfor capturing a single particle and is similar to trap 746 of FIG. 22,described above, except that trap 858 disposes channel 862 againstdistal wall 864, in contrast to trap 746, which spaces channel 752 awayfrom distal wall 716.

[0489] Particle retention and treatment are essentially as described forExample 2 above, but the operation of a slightly different control layer866 is described here for clarity. Control layer 866 includes valvesV1-V4. Valve V1 corresponds to valve 8 of FIG. 8, described above, andis used to convert between divided and unified flow paths. Valve V2controls particle release mechanism 852; its function is describedbelow. Valves V3 and V4 control fluidic flow to waste reservoir 868 andparticle collection mechanism 854, respectively. During particle loadinginto trap 858, valves V1, V2, and V3 are open, and valve V4 is closed.During reagent delivery by perfusion mechanism 860, valves V1 and V4 areclosed, and valves V2 and V3 are open.

[0490] Particle release mechanism 852 may be used at any time to releaseparticles, particularly after use of perfusion mechanism 860 and/ormeasurement of trapped particles. Release mechanism 852 operates by adislodging flow to propel retained particles out their confinement intrap 858; see FIGS. 24 and 25. The dislodging flow originates in areservoir channel 870 that is fluidically connected to trap 858 using asize-selective channel 872. Size-selective channel 872 has a diameterthat prevents entry of particles but that does not restrict passage offluid to, or from, reservoir channel 870.

[0491] Fluid flow through size-selective channel 872, and thus particlerelease, is controlled by valve V2 (see FIG. 24). Valve V2 is acontrol-layer valve disposed over reservoir channel 870. When valve V2is closed, reservoir channel is compressed, forcing fluid outwardthrough size-selective channel 872 into trap 858. This releases trappedparticles, propelling them out of trap 858 into a flow stream, such asmain flow stream 874, shown in FIG. 25, which carries the particles awayfrom trap 858. Typically, in use, the focusing buffer pump is running,the reagent valves are closed, and the shield buffer is running. Thus,the main flow stream goes from the buffer wells to the cell culturearea, described below. When valve V2 is opened, reservoir channel 870expands, bringing fluid in through size-selective channel 868 andrefilling the reservoir channel.

[0492] Embodiment 2

[0493]FIG. 26 shows a system 880 for retaining and releasing groups ofparticles, in accordance with aspects of the invention. System 880generally is similar to system 850 (compare with FIG. 25), but withseveral exceptions. First, trap 882 includes a much larger retentionsite 884 than trap 858, capable of holding a group of particles. Thus,walls 886 extend substantially into cross channel 888, and each wallincludes three size-selective channels 890, rather than the one presentin trap 858. Moreover, trap 882 is wider than trap 858, so multipleexpulsion channels 892 are used to release particles from confinement intrap 882, rather than one. Second, perfusion channel 894 has been movedslightly away from focusing channel 896 to ensure effective delivery ofreagents to all particles in trap 882.

[0494] Released particles generally may be discarded or saved forfurther treatment and/or analysis, for any trap size or configuration.Particles to be discarded may be carried toward waste reservoir 868 byopening valve V3 and closing valves V1 and V4 (see FIG. 24).Alternatively, particles to be saved may be carried toward particlecollection mechanism 854 by opening valve V4 and closing valves V3 andV1 during particle release. Thus, valves V3 and V4 provide a sortingmechanism 898 to selectively discard or collect each individual particleor group of particles.

[0495] Once a retained particle has been released, system 850 may bereadied to trap another particle. Toward this end, valve V4 is closed,if it was opened during particle release, and valves V1, V2, and V3 areopened. System 850 then is ready to receive another particle.

[0496] Embodiment 3

[0497]FIGS. 24 and 27 show a particle collection mechanism 854, inaccordance with aspects of the invention. Collection mechanism 854includes an inlet channel 904, a retention area 906, filter channels908, and an outlet 910. Inlet channel 904 carries released particlestoward retention area 906 when valve V4 is open during release. Fluidflows through retention area 906 to outlet 910 by passing through filterchannels 908, which act as size-selective channels that prevent releasedparticles from flowing to the outlet. Thus, released particle arecollected in retention area 906. When the collected particles are cells,the retention area may be used to culture cells to promote cell growth,differentiation, and/or response to a treatment, such as by perfusionmechanism 860. Alternatively, the retention area may be operativelyconnected to a measurement system for particle analysis, and/or may be asite of particle lysis or further treatment. In some embodiments, inletchannel 904 may be connected to other channels (not shown) that allowreagents to be introduced to retention area 906 separate from particleretention, treatment, and analysis at trap 858. Alternatively, or inaddition, reagents may be introduced by perfusion mechanism 860 and/orfocusing channel 896. Particles collected in retention area 906 may bereleased by reversed flow to send them up inlet channel 904 or byforming collection mechanism 854 so that a valve (or valves) replacessome of the filter channels.

[0498] Embodiment 4

[0499] Standard particle input mechanisms, such as inlet-well 330 ofFIG. 8, are sufficient for single-use microfluidic systems. However,these mechanisms may be inadequate for reusable systems. In reusablesystems, it may be desirable to load a suspension of particles into aninlet-reservoir(s) at the beginning of an analysis, and then to use thatsame suspension as a source for multiple particle loadings and analyses.Unfortunately, during such extended analyses, particles typically settleout of the suspension, so that the particle input concentrationdecreases with time, increasing the amount of time required to loadparticles. Thus, there is a need for a mechanism for maintainingparticles in suspension in an inlet reservoir during extended analyses,to allow repeated loading and analysis of particles from thissuspension.

[0500]FIG. 28 shows a particle suspension mechanism 920 that may beintegrated into reusable microfluidic systems, such as systems 850 and880 described above. This suspension mechanism helps to maintainparticles in suspension and/or helps to resuspend settled particlesduring the course of analyses with a reusable microfluidic system.Mechanism 920 includes an inlet reservoir 922, recirculation channels924, and pumping valves 926. Inlet reservoir 922 receives and storesparticle suspensions during analyses. Thus, reservoir 922 may be aninterface with the macroscopic world. Recirculation channels 924 arejoined at each end 928 to the base of reservoir, but are spaced from thereservoir at an intermediate portion 930. Pumping valves 926 areregulated by the control layer, and are coordinated to peristalticallypump fluid through recirculation channels 924, as described elsewhere inthis Detailed Description. Accordingly, fluid in reservoir 922 flowsaway from, and then back to, reservoir 922, continuously acting to mixthe contents of reservoir 922 and thus to maintain the particles insuspension. Therefore, a more stable concentration of particle flowsfrom outlet 932 over time.

Example 8 Microfluidic Mechanisms for Adjustable Reagent Delivery

[0501] This example describes mechanisms for adjustably dilutingreagents so that reagents may be delivered to particles at a range ofreagent concentrations, for example, as a gradient; see FIGS. 29-30.

[0502] Background

[0503] Studies of cells frequently involve dose-response analyses todetermine how the cells respond to a range of concentrations of areagent, such as a drug. These dose-response analyses may be used todetermine a variety of qualitative and/or quantitative information,including an effective dose, a half-maximal response dose, a lethaldose, a dose to produce a more specific response, and so on. In manyanalyses, a reagent of interest is prepared as a high concentrationstock solution, and then various volumes of the reagent are dispensed toprovide a range of doses. However, this approach may not be suitablewith microfluidic systems, because it may not be practical to dispensemetered volumes in a microfluidic system and because it may require amixer to mix and thus dilute such a dispensed volume. Thus, there is aneed for a microfluidic mechanism that dispenses a premixed reagent at arange of selected concentrations, using a small number of reagentstocks.

[0504] Description

[0505] This section describes two exemplary dilution mechanisms, havingindependent (Embodiment 1) and coordinated (Embodiment 2) control.

[0506] Embodiment 1

[0507]FIG. 29 shows an adjustable dilution mechanism 960 for combiningfirst and second reagents at a range of concentrations, in accordancewith aspects of the invention. Dilution mechanism 960 includes amicrofluidic layer 962 having first and second reagent reservoirs 964,966, and first and second controllable flow channels 968, 970 acting asoutlets for the reservoirs. The controllable flow channels narrow andmeet at a junction 972 to form a common mixing channel 974. Reagents aremixed in mixing or diffusion channel 974, generally by diffusion ofreagents into the adjacent flow stream(s). Thus, mixing channel 974 maybe substantially narrower than flow channels 968, 970, generally about 1to 20 μm. In contrast, flow channels 968, 970 are wide enough to becontrolled by valves, with an arcuate cross-section. Here, fluid flowfrom each reservoir is independently controlled by control layer 976,via three-valve pumps 978, and shutoff valves 980; however, fluid flowin other embodiments may be controlled by other control mechanisms.

[0508] Dilution mechanism 960 is used to combine first and secondreagents, R1 and R2, in a desired ratio based on the rate at which eachpump moves fluid through flow channels 968, 970. Thus, reagent R1 may beintroduced, for example, at 100%, 50%, 20%, 10% and 0% of reservoir 964concentration, by running pumps 976 and 978 at relative pumping flowrates of 1:0, 1:1, 1:4, 1:9, and 0:1, respectively. Valves 980 may beused to override the pump and/or to modulate the effect of a specificpump rate, as described below. To improve control, the adjustabledilution mechanism may use relatively precise control of pump speed anda large number of control lines in the control layer.

[0509] Embodiment 2

[0510]FIG. 30 shows another adjustable dilution mechanism 990 forcombining first and second reagents at a range of concentrations, inaccordance with aspects of the invention. Dilution mechanism 990 isstructured similarly to dilution mechanism 960, as indicated bycomponents with identical numbering. However, dilution mechanism 990uses a single pump 978, generally at a constant pumping rate, tocoordinately drive flow of both reagents. Furthermore, mechanism 990uses adjustable valves 994, 996, rather than shutoff valves. Closure ofadjustable valves is controllable by regulating the pressure used todeflect the adjustable valves. Thus, each adjustable valve may beindependently adjusted with a suitable pressure to provide a desiredpartial obstruction to flow channels 968, 970, and thus a desired flowrate and reagent mixture in diffusion channel 974. A simple dilution ofa first reagent may be carried out by using an appropriate solvent orbuffer as the second reagent.

[0511] Applications

[0512] The dilution mechanisms described above may be used as part(s) ofany suitable microfluidic device, for any suitable applications. Forexample, dilution mechanism 990 may be used in microfluidic system 250in FIG. 8 of Example 2 to prepare and deliver a desired mixture ofreagents for particle perfusion, by providing empirically determinedpressures to valves 9 and 10.

Example 9 Microfluidic Sorting Mechanisms Based on Centrifugal Forces

[0513] This example describes mechanisms for sorting particles based ontheir mass, density, and/or other properties; see FIGS. 31-38.

[0514] Background

[0515] Microfluidic analyses of particles may benefit from or evenrequire sorting crude or heterogeneous input populations of particlesinto their components. For example, the input population may be amixture of single cells, cell clusters, and/or cell debris.Alternatively, or in addition, the input population may be a mixedpopulation of distinct cell types. In these cases, sorting may separatesingle cells from clusters and debris, and cells of one type from cellsof another type. Optical systems may be used to actively sort individualparticles according to their different optical properties, such asfluorescence intensity. However, these optical systems require that theinput particles be constantly monitored and actively directed todistinct sorting bins based on optical properties. Thus, there is a needfor a microfluidic sorting mechanism that separates distinct particles,potentially passively, based on different physical properties of thedistinct particles.

[0516] Description

[0517] This example describes mechanisms for passively sorting particlesbased on physical differences between the particles, such as mass,density, shape, and/or surface characteristics, among others. Thesemechanisms are passive, exploiting the centrifugal forces exerted onflowing particles during a sharp change of direction, rather than activemonitoring and switching. These mechanisms are described anddemonstrated as part of simplified fluidic systems lacking valves andother functional mechanisms. Instead, fluids are moved through thesesystems by pressure differences produced by liquid columns havingdifferent heights in input and output reservoirs. However, these sortingmechanisms may be integrated into any suitable microfluidic system.

[0518] Embodiment 1

[0519]FIGS. 31 and 32 show a microfluidic system 1020 having a sortingmechanism 1022 that separates particles according to physicaldifferences between the particles, in accordance with aspects of theinvention. Here, mechanism 1022 sorts particles from inlet reservoir1024 into one of three outlet or sorting channels 1026. These sortingchannels lead to distinct outlet reservoirs 1028, labeled here asoutlets 1-3. The sorting channels in this embodiment have a minimumwidth of about 50 μm and a height of about 17-18 μm. However, moregenerally, mechanism 1022 may be formed with any suitable dimensions.Furthermore, mechanism 1022 may sort particles from any suitable source,such as a microfluidic treatment or analysis, into any desired number ofoutlet channels and/or other microfluidic mechanisms or structures, suchas culture chambers, retention mechanisms, perfusion mechanisms, and/orthe like.

[0520] Mechanism 1022 includes structures that act sequentially along aflow stream. First, hydrodynamic focusing region 1030 acts to focusparticles from particle inlet channel 1032 into a narrow stream. Twoside reservoirs 1034, 1036, each filled with a focusing fluid, such as abuffer, are connected to inlet channel 1032 using focusing channels1038, 1040. Focusing channels 1038, 1040 may have different widths, andthus different flow rates, to asymmetrically position the narrow streamin the inlet channel. Second, acceleration region 1042 narrows the widthof the channel to increase the flow velocity and further focus particlesinto a single stream. Third, curved region 1044 bends sharply to givethe input particles an angular velocity and a radial acceleration.Fourth, a separation region 1046 is positioned after curved region 1044.Separation region 1046 widens into a larger chamber with a number ofreceiving or sorting channels 1026 that act as sorting bins to segregatesorted particles. In separation region 1046, particles are distributedbased on their mass (weight). The tendency of particles to continuemoving in a straight line increases with mass, so that heavier particlesmove to the outside of the flow stream, and lighter particles remaincloser to the center of the flow stream. Accordingly, in thisembodiment, the heaviest particles tend to distribute more to receivingchannel 1048, the lightest particles to receiving channel 1050, and theintermediate-mass particles to receiving channel 1052. In some cases,other physical properties of the particles, such as density, shape,and/or surface properties, among others, also may contribute to therelative distributions of particles between these receiving channels.

[0521] The sorting capabilities of sorting mechanism 1022 may bemodified by altering one or more of several potential sortingparameters. These sorting parameters may include the extent of narrowingof the acceleration region, the radius of curvature of the curvedregion, the angle of broadening of the separation region, and/or thenumber of receiving channels/bins, among others. These parameters mayimpart such capabilities as improved resolution, separation into adifferent number of sorting channels (bins) and/or resolution of adifferent range of particle weights, densities, etc.; among others.

[0522] Embodiment 2

[0523]FIG. 33 shows a microfluidic system 1060 having a sortingmechanism 1062 with modified sorting parameters, in accordance withaspects of the invention. Sorting mechanism 1062 has a narroweracceleration region 1064 than acceleration region 1042 of sortingmechanism 1022, potentially imparting greater velocity to the particles,and thus better focusing. In addition, sorting mechanism 1062 has acurved region 1066 with a distinct radius of curvature relative tocurved region 1044 of sorting mechanism 1022. Furthermore, sortingmechanism 1062 has a separation region 1068 having a greater angle ofseparation (subtended angle) than separation region 1046 of sortingmechanism 1022, connected to four, rather than three, sorting channels1070.

[0524] Embodiment 3

[0525]FIGS. 34 and 35 show another microfluidic system 1080 having asorting mechanism 1082 with modified sorting parameters, in accordancewith aspects of the invention. Sorting mechanism 1082 has a narroweracceleration region 1084 than either region 1042 or region 1064,providing even greater velocity and focusing. In addition, sortingmechanism 1082 has a curved region 1086 with a smaller radius ofcurvature than curved regions 1044 and 1066 of FIGS. 31-33. Furthermore,sorting mechanism 1082 has a separation region 1088 with an even greaterangle of separation, compared to regions 1046 and 1068.

[0526] Applications

[0527] FIGS. 36-38 show experimental results demonstrating the abilityof systems 1020 and 1060 to sort a mixed population of particles. Inthese experiments, the mixed population of particles was formed, priorto loading into an input reservoir, using two sizes (and types) ofparticles: beads with an average diameter of about 1 μm, and Jurkatcells with an average diameter of about 10 μm. These two sizes ofparticles are distinguishably labeled with distinct fluorescent dyes:the beads emit green light, and the cells emit red light.

[0528]FIG. 36 shows an image of particles being sorted using a sortingmechanism as described in this example. The particles are split into twostreams 1100 in the separation region. The lower stream is enriched forcells (red), and the upper stream is enriched for beads (green). Flow ofparticles through the system is powered by a 1-cm high column of fluidin the inlet reservoir.

[0529]FIGS. 37 and 38 show graphs of data obtained with systems 1080 and1020, respectively, as each sorted the mixed population of beads andcells, described above. These graphs were generated by counting therelative numbers of particles that entered each of two receivingchannels. The graphs each plot the fraction of cells (blue diamonds) andbeads (pink squares) that distribute to the lower receiving channel,either sorting channel 1102 or 1048, respectively. The ratio of cells tobeads in the lower receiving channel is plotted in yellow. In bothsystem 1080 and 1020, a greater fraction of cells than beads areentering the lower receiving channel. In system 1080, about twice asmany cells as beads entered the lower receiving channel. In system 1020,this ratio was slightly lower and more variable.

[0530] Summary

[0531] The systems shown in this example have the ability to passivelyenrich particles based on sorting mechanisms that distinguish physicalproperties of particles. The approximately two-fold enrichment obtainedusing these systems may be sufficient to facilitate or improve somemicrofluidic analyses. Furthermore, each of these systems may bemodified and refined, and/or connected in series to improve enrichmentof desired particles.

Example 10 Microfluidic Systems for Manipulating Sets of Particles

[0532] This example describes microfluidic systems having relativelylarge chambers, in which larger sets of particles, such as adherentand/or nonadherent cells, can be retained, stored, cultured, treated,and/or released; see FIGS. 39-50D.

[0533] Background

[0534] The introduction and/or removal of particles into and out ofmicrofluidic systems, at macroscopic/microscopic interfaces, mayinefficient and/or harmful. For introduction, particles must be placedin suspension and often are introduced through an inlet reservoir.During this loading process, a substantial fraction of the particles maybe lost, which may be problematic if the particles are expensive and/orin limited supply, such as with cells from a clinical or forensicsample. Furthermore, during introduction and/or removal, particles maybe contaminated, for example, by exposure to contaminatingmicroorganisms, and/or damaged, for example, by evaporation of inlet- oroutlet-reservoir liquid. Accordingly, it is desirable to avoidrepeatedly introducing and removing particles from microfluidic systemsduring a sequential set of assays. Therefore, there is a need forchambers for storing, treating, maintaining, measuring, and/or inparticular, amplifying (i.e., culturing) particles, such as cells,particularly for serial analyses of particle populations. With suchchambers, these serial analyses could be conducted without transferringthe populations to a macroscopic environment between analyses.

[0535] However, such chambers need to address a number of problems orissues related to their use with cells. First, these chambers may need aceiling height that does not interfere with cell movement within thechambers. In particular, the ceiling of larger chambers, particularlythose formed of elastomeric materials, may tend to sag, obstructing cellmovement. Second, these chambers may need a substrate that promotesadhesion, survival, and growth of adherent cells, when such cells arebeing used. Many adherent cells do not behave normally unless they areattached to a substrate. Third these chambers may need to pass mediaand/or reagents over cells in the chambers, without loss of, or damageto, the cells. Pumps that circulate fluid may crush fragile eukaryoticcells, and some filters that restrict cell movement may be clogged bycells and/or allow cells to pass. Fourth, these chambers may require anability for gas to diffuse into cell chambers, to maintain a proper pHduring cell growth.

[0536] Description

[0537] This example describes various microfluidic systems that addressand solve some or all of the problems and issues cited above. Thesemicrofluidic systems may be formed using multilayer soft lithography, asdescribed elsewhere in this Detailed Description and in theCross-References. Channels or chambers for particle storage, treatment,analysis, and cell growth are formed using molds fabricated as describedgenerally in Example 13, using plural layers of photoresist, whenneeded. Such molds may be used to construct channels large enough forcell entry and growth, for example, about 200 μm wide by about 20-35 μmhigh. Furthermore, as described below, such molds may be used to formparticle chambers of various dimensions. These channels and/or chambersmay be integrated into microfluidic systems that include valves, pumps,rotary mixers, filters, sorters, multiplexers, perfusion mechanisms,and/or additional particle retention sites, among others, to perform anysuitable analysis of particles.

[0538] Embodiment 1

[0539] FIGS. 39-43 illustrate exemplary microfluidic networks 1130 thatinclude relatively large chambers 1132 for retaining particles, inaccordance with aspects of the invention. These networks have beenfabricated using multilayer soft lithography, with large chambers thatdid not collapse. These chambers have a height of about 36 microns. Thechambers were formed by a modified process using molds in which twolayers, each of about 18 microns, were sequentially layered on top of asubstrate, and selectively retained at the positions where the cellchambers were formed. The chambers were rounded. This process produces agenerally arcuate (arch-like) cross-sectional configuration that mayenhance stability. As a result, this process allows formation ofchambers with width-to-height ratios less than about 10:1 that do notcollapse. In contrast, microfluidic channels having width-to-heightratios greater than 10:1 formed by a standard soft lithography processmay collapse more frequently.

[0540] The large chambers may be connected to an input reservoir 1134and an output reservoir 1136. The input reservoir may connect to aninlet channel 1138 that bifurcates, as shown at 1140, to direct flowinto each of two channels 1142. Outlet channels 1144 extend from eachpair of chambers to join and carry fluid to output reservoir 1136. Formore efficient use of space and input reservoirs, some systems, such assystem 1146, share a common inlet reservoir 1148 for two pairs ofchambers. Thus, particles may be loaded into inlet reservoir 1148 todistribute the particles to each of four chambers. In other embodiments,an input reservoir may be fluidically connected to one, two, three,four, or more chambers using any suitable number of channels. Thechannels may extend directly between a particle reservoir and a cellchamber, or they may branch any desired number of times at any desirednumber of positions. The movement of fluid through these chambers may becontrolled by any suitable mechanism, such as valves and/or pumps, amongothers. For example, FIG. 44 shows a system 1150, in which an array ofnetworks 1130 are controlled in parallel by control lines 1152, 1154that regulate valves 1156 flanking each chamber 1132. In this case, eachof the eight valves shown is opened or closed in parallel throughactuation at control port 1158, either providing an open chamber forparticle loading, or a closed chamber for particle isolation,respectively.

[0541] Chambers 1132 may have any desired shape and size. Suitablecross-sectional shapes may include diamonds 1160 (FIGS. 39 and 41),rectangles 11-62 (FIGS. 39, 42, and 43), squares 1164 (FIG. 39), circles1166 (FIGS. 39 and 40), ellipses or elongated circles 1168 (FIGS. 39,40, and 41), and/or the like. Suitable sizes are about 100 microns toabout 1 centimeter in diameter, depending on particle type, assay, andso on. Specific chambers shown in FIGS. 39-43 that have been constructedsuccessfully have diameters of from about 0.9 mm to 2.6 mm.

[0542] Chambers may be completely isolated from the substrate in theirinteriors, or they may be supported by columns, posts, or otherstructures. These columns or posts may project downward from the roof ofthe channel to contact the substrate, generally being integrally formedin the microfluidic layer during fabrication of this layer.Alternatively, or in addition, these columns or posts may project upwardfrom the substrate, being formed as a portion of the substrate or anaddition to the substrate. To be effective, the columns or posts shouldbe spaced adequately to avoid obstructing cell movement through thechambers, although more tightly spaced structures could be used to forma cell pen or other subchamber.

[0543] Embodiment 2

[0544]FIG. 45 shows a microfluidic system 1180 having a microfluidicnetwork 1130 through which fluid flow is more flexibly controlled.Specifically, fluid flow through chamber 1132 is controllable by twonested sets of flanking control valves 1182, 1184 that sit to both sidesof chamber 1132. A parallel pumping circuit 1186 is disposed as anparallel fluid path 1188, having pump 1190 and extending from upstreamand downstream cell chamber 1132, at an intermediate nested-positionbetween nested valve sets 1182, 1184.

[0545] System 1180 may be operated as follows. During cell (particle)loading, nested valve sets 1182, 1184 are opened and fluid flowspassively from input reservoir 1134 to output reservoir 1136, bringingcells to chamber 1132. When a desired number of cells have enteredchamber 1132, one or both of valve sets 1182, 1184 are closed to isolatechamber 1132. If only valve set 1182 is closed, pump 1190 may beactivated to circulate fluid through a loop that include chamber 1132and alternate fluid path 1188, to prevent cell adhesion to thesubstrate, or to maintain a fluid flow over cells that have adhered.Alternatively, only valve set 1184 may be closed, allowing fluid to flowbetween input and output reservoirs using alternate, parallel fluid path1188, to the exclusion of a path through chamber 1132. Thus, fluidchannels may be flushed and re-equilibrated with any desired reagent.Once the fluid channels have been re-equilibrated, the desired reagent,valve set 1182 may be closed and the desired valve set 1184 may beopened, to actively pump the desired reagent in a closed loop thatincludes chamber 1132. For example, the reagent may be a mixture oftrypsin and EDTA, or another suitable detaching reagent. Pumping themixture of trypsin and EDTA through the closed loop detaches adheredcells. Opening valve set 1182 then allows the detached cells to beflushed from the system, either to output reservoir 1136 or to anyadditional microfluidic mechanism or set of mechanisms, as describedthroughout this detailed description.

[0546] Embodiment 3

[0547]FIG. 46 shows a microfluidic system 1210 with a cell chamber 1212formed as a looped channel or ring structure, in accordance with aspectsof the invention. Cells (or particles) are introduced into chamber 1212and retained there, either by balancing fluid height between input andoutput reservoir 1214, 1216, respectively, or by closing one or morevalves 1218 that interconnect these reservoirs. Partial closure ofvalves 1218, particularly valves adjacent or within chamber 1212, may beused to permit fluid flow, while preventing cell flow, past the valves.Once cells are loaded into chamber 1212, four valves 1220 may beactuated in an appropriate order to move fluid around chamber 1212

[0548] Embodiment 4

[0549] FIGS. 47-49 shows another microfluidic system 1240 with a chamber1242 formed as a looped channel or ring structure, in accordance withaspects of the invention. System 1240 offers distinct networks forparticle inflow/outflow—particle network 1244—and for reagentinflow/outflow—reagent network 1246. These distinct networks overlap atchamber 1242.

[0550] Particle network 1244 is used to load particles into chamber 1242and to receive particles flowing from chamber 1242. Particles are loadedinitially into input reservoir 1248, which feeds the particles intoinput channel 1250. Input channel 1250 flows into chamber 1242 Chamber1242 bifurcates and rejoins at outlet channel 1252. Outlet channel 1252carries fluid to output reservoir 1254. Fluid flow between reservoirs1248 and 1254 can be terminated at any selected time by closing one orboth of valves 1256 and 1258. Closing both valves fluidically isolateschamber 1242 from the remainder of particle network 1244.

[0551] Reagent network 1246 is used to move fluid, particularly fluidcarrying reagents, through chamber 1242, while selectively retainingparticles. Reagent network 1246 directs fluid and reagents from one ormore reagent reservoirs 1260 through inlet channel 1262 into chamber1242. Flow from each reagent reservoir 1260 is independently regulatedby valves 1264, which control flow of a single reagent or a mixture ofreagents. Desired ratios and/or dilutions of reagents may be formed byprecisely controlling flow rate through each valve, for example, asdescribed above in Example 8. Reagents entering chamber 1242 from inletchannel 1262 follow a bifurcated path that rejoins at outlet channel1266. Outlet channel 1266 carries fluid to waste reservoir 1268. Inflowor outflow can be regulated with valves 1270, 1272, respectively, whichmay be closed to isolate chamber 1242 from reagent network 1246,particularly during particle loading and/or removal. Furthermore, areagent pump 1274 may be used to pull reagents from reagent reservoirs1260 to waste reservoir 1268.

[0552] Reagent network 1246 blocks exit (and entry) of particles from(and to) chamber 1242, based on particle size. To achieve this, reagentnetwork 1246 interfaces with chamber 1242 using filtering mechanisms1276. FIGS. 48 and 49 show photographs of size-selective channels 1278disposed in outlet channel 1266, adjacent chamber 1242.

[0553] Chamber 1242 includes a chamber pump 1280 (see FIG. 47). Chamberpump 1280 is used to circulate fluid through chamber 1242, for example,(1) to suspend cells (such as during detachment of adhered cells withtrypsin), (2) to move cells away from filtering mechanism 1276, reducingor preventing clogging of the mechanism, (3) to promote mixing withinchamber 1242, and/or the like.

[0554] An exemplary method for feeding cells in chamber 1242 is afollows. One of reagent reservoirs 1260 is loaded with about 20 μLmedia, and waste reservoir 1268 is loaded with about 10 μL media (orbuffer). These reservoirs have the same diameter, so this asymmetricalloading gives reagent reservoir 1260 a fluid head of about 10 μL. Flowto equalize fluid heights subsequently transfers about 5 μL of mediathrough chamber 1242 to waste reservoir 1268 over the course of about 30min. Particle network 1244 may be used instead, or in addition, if thecells in chamber 1242 are adherent.

[0555] System 1240 allows extended culture of adherent cells. FIG. 50shows NIH 3T3 cells 1290 that are alive and adherent in chamber 1242, 3weeks after they were seeded. The field of cells shown has been testedfor viability (top panel) and visualized for general morphology bybright field illumination (bottom panel). A substantial majority ofcells was determined to be alive, as evidenced by lack of ethidiumhomodimer staining (Molecular Probes; Live/Dead Viability Assay Kit),and to have normal morphology. During the 3-week incubation, cells 1290were subjected to the passive-flow feeding regimen described above,repeated once every 2 days.

[0556] Embodiment 5

[0557]FIG. 50A shows a system 1910 for depositing cells (or otherparticles) in a microfluidic chamber 1912, based on an asymmetricallydisposed flow path. Particles and fluid flow into chamber 1912 frominlet channel 1914. The particles and fluid may follow plural distinctflow paths 1916, 1918 toward outlet channels 1920, 1922, respectively.One or more valves 1924 may be used to select one or both of the flowpaths.

[0558] Selection of asymmetrically disposed flow path 1916 allows asubset of inputted cells to be deposited in chamber 1912. Main flow path1916 may be both asymmetrically disposed and nonlinear. Such a flow pathdefines a highest velocity main stream corresponding to main flow path1916. However, some of the fluid also follows lower-velocity auxiliarystreams (weaker flow streams) disposed more distally in chamber 1912, inquasi-stagnant region 1926. Accordingly, the subset of cells thatfollows the auxiliary streams within chamber 1912 tend to be depositedin chamber 1912 by settling out and contacting a substrate defined bythe chamber. Such contact diminishes the ability of fluid flow to movethe settled cells and may promote additional interactions between thesettled cells and the substrate, such as formation of a secretedextracellular matrix. In other embodiments, the subset of cells that aredeposited may be determined by varying any suitable parameters includingdegree of nonlinearity of flow path 1916, location of flow path 1916relative to the chamber, chamber dimensions, fluid flow rate, and/or thelike.

[0559] Embodiment 6

[0560]FIG. 50B shows a system 1930 that is based on system 1910 butincludes additional mechanisms and features. System 1930 includes aninput mechanism 1932, an output mechanism 1934, and a treatmentmechanism 1936. Input mechanism 1932 includes an input reservoir 1938for introducing cells and/or fluid, such as buffer or media. Outputmechanism 1934 includes an output reservoir 1940 that may receive fluidfrom outlet channels 1942 and/or 1944, provided by flow paths 1918and/or 1916, respectively. Valve 1924 may be operated to block flowalong path 1918, whereas valve 1948 may be operated to block flow tooutput reservoir 1940 from either flow path. Treatment mechanism 1936may include plural reagent reservoirs 1950, valves 1952 that regulateflow from each reagent reservoir, and a valve 1954 to regulatecommunication between entire treatment mechanism 1936 and chamber 1912.

[0561] System 1930 may be used to deposit cells as follows. Cells areinputted by input mechanism 1932, generally with valve 1948 opened, andvalve 1924 closed. Cells travel along flow path 1916, with a subsetfollowing auxiliary flow streams to be deposited in quasi-stagnantregion 1926, as described above.

[0562] Once a sufficient number of cells have been deposited withinchamber 1912, the deposited cells may be manipulated further as follows.Valve 1956 may be closed and the contents of input reservoir 1938replaced with media to achieve a fluid head that is approximately equalto that of output reservoir 1940, to produce no net flow betweenreservoirs (a “balanced flow” condition), and then valve 1956 may bereopened. The deposited cells may be incubated a suitable time period,such as overnight, during which time they may adhere by interaction witha substrate defined by the chamber. Such adhered cells are retainedwithin chamber 1926. Alternatively, nonadherent cells may be usedwithout attachment to chamber 1912.

[0563] Adhered (or nonadhered) cells may be treated with reagents fromreagent reservoirs 1950 by operating treatment mechanism 1936. First,reagents may be introduced into chamber 1912 by opening one or morevalves 1952, and valve 1954, to direct selected reagents along flow path1958, along a reverse of flow path 1916, and/or along outlet channel1944. Next, chamber 1912 may be placed within a closed loop by closingvalves 1948, 1954, and 1956. Pump 1960 may be started to circulatereagent around the closed loop, providing a mixing action thatcontinuously perfuses cells in chamber 1912 with reagent.

[0564] Embodiment 7

[0565]FIG. 50C shows a cell chamber 1970 that may be used to deposit(and retain) cells in one or two compartments 1972, 1974. Compartments1972, 1974 may be connected by radially arrayed, size-selective channels1976 to form a “spoked wheel” structure. Cells (or other particles) maybe inputted from first input channel 1978 and deposited in compartment1972. Fluid may flow through size-selective channels 1976 to secondinput channel 1980. Alternatively, or in addition, additional cells,such as a distinct cell type, may be inputted from second input channel1980 to be deposited in outer compartment 1974, with fluid flowingtoward first input channel 1978. With each of the two compartmentsoccupied by distinct cell populations, cell-cell communication may beanalyzed by passage of released cell components (or extended cellstructures) through the size-selective channels between the twocompartments. In alternative embodiments, the first and secondcompartments may have any suitable geometry, such as interdigitatedfingers or intermeshed spirals, among others, to increase the area ofcommunication between the two compartments. Furthermore, additionalcompartments may be added to measure interactions between additionalcell types.

[0566] Embodiment 8

[0567] Cell chamber 1990 is a modified version of chamber 1970 thatincludes an overflow capability. Here, inner compartment 1972 acts as achamber that is connected to overflow compartment 1992 by transversepassages 1994, in addition to size-selective channels 1976. Accordingly,input channel 1978 may be used to direct most of inputted cells (orother particles) into inner compartment 1972 using entrance 1996.However, once inner compartment 1972 becomes filled, additional cellsmay travel along transverse passages, through overflow compartment 1992and out outlet channel 1998.

[0568] Applications

[0569] The microfluidic systems described here may be used for themanipulation of adherent and nonadherent cells. For example, afterintroduction to a chamber, NIH 3T3 cells adhere to the substrate toretain the cells effectively within the chamber. Once adhered, thesecells remain attached to the substrate as fluidic flows are directedover them passively and/or actively. These cells remain viable at arange of flow rates and valve closure pressures. However, cell viabilitymay be compromised when higher valve actuation pressures are used,because higher pressures lead to complete valve closure. A valve thatcloses upon a cell can crush it. In particular, at high pumpingfrequencies, all cells within a population inside a ring may be crushed,since they have a high probability of being crushed. In this case, thering may become filled with cell debris, which may be a starting pointfor assays on cell components. The nuclear membrane may or may not becompromised by this treatment.

[0570] In general, manipulation of adherent cells on the chips isachieved in the following manner. Adherent cells are prepared from seedflasks by releasing the cells from the flasks, for example, bytrypsinization, followed by washing, centrifugation, and resuspension ina standard tissue culture medium, such as DMEM or RPMI. Once a desiredconcentration has been achieved, cells are loaded using a manualpipettor into the input well and cells flow into the microfluidicchannel structures under the head flow generated by the column ofliquid. Once adhered, adherent cells can be resuspended in themicrofluidic channel by addition of trypsin-EDTA or other cell-detachingagents.

[0571] The microfluidic layer and substrate may be treated (or leftuntreated) to promote cell flow, cell viability, cell adhesion ornonadhesion, cell growth, and/or the like. Fluidic channels and/or thesubstrate may be treated with a nonionic detergent, such as TWEEN; aserum protein, such as a serum albumin (e.g., BSA); whole orfractionated serum from any suitable animal; extracellular matrixextracts, components, or mixtures, such as collagen, polylysine,SIGMACOTE, MATRIGEL, etc.; and/or the like.

Example 11 Systems for Electrophysiological Analysis of Cells in aMicrofluidic Environment

[0572] This example describes microfluidic systems for positioning,retaining, treating, and/or measuring cells, particularly forelectrophysiological analyses; see FIGS. 51-58.

[0573] Background

[0574] Cell-surface membranes are an essential part of all cells,defining their extent, and separating and maintaining the differencesbetween the cell interior (cytoplasm) and the extracellular milieu.Accordingly, controlling membrane permeability and the selectivity ofion movement across membranes, mediated by ion channels andtransporters, is fundamental to cell survival, cell physiology, andsignal transduction mechanisms, particularly neurotransduction. Thus,many cell-surface receptors couple to ion channels and transporters,making measurement of membrane currents a very rapid and sensitiveindicator of cell physiology and receptor activity. Therefore, many drugassays benefit from or, in some cases, require a measurement of theeffects of drugs on ion currents, referred to as electrophysiology.

[0575] The preferred method for conducting electrophysiological analysesof cells membranes is the “patch-clamp” analysis of individual cells.Typically, in this approach, a glass electrode with a diameter of about0.1-1 μm is electrically sealed against the membrane of a single cell,surrounding a membrane “patch” on the cell. The patch then may be leftintact, separated from the cell, “perforated” with channel-formingagents, or penetrated, based on the type of information desired. Withboth intact patches and patches separated from a cell, the size of thepatch and the density of channels in the membrane determine the numberof channels being analyzed. Thus, different sizes of patches may allow“single-channel recordings” from small regions of membrane, orrecordings from many of channels in “macropatch recording.”Alternatively, membrane patches can be perforated or penetrated tomeasure electrical properties of the entire cell membrane, in“whole-cell” patch-clamp studies. Perforated patches introduce achannel-forming agent, such as the antibiotics nystatin or amphotericinB, into the membrane. Perforated patches enable whole cell recording ofchannel activity with loss of larger cytoplasmic components. Penetratedpatches place an electrode inside a cell, so that the electrode and thecell's cytoplasm are continuous. Accordingly, penetrated patches alsoenable whole-cell patch-clamp recording.

[0576] Despite the importance of electrophysiology as an assay tool andthe variety of patch-clamp methods available for measuring electricalactivity at membranes, these methods require substantial time and skillfor their proper execution. In particular, each of these methodsgenerally is carried out manually, by a highly-skilledelectrophysiologist. The electrophysiologist must precisely position anelectrode against the membrane of each cell, and manipulate theelectrode and/or cell additionally to form a gigaseal and/or penetratethe cell. Accordingly, the electrophysiologist must devote considerabletime and energy to the execution of patch-clamp methods, making themexpensive and ill-suited to screening applications in which many samplesmust be studied. Thus, there is a need for a more automated system thatsimplifies cell manipulation and at least partially automates patchformation.

[0577] Description

[0578] This example describes microfluidic devices that allowmeasurements of ion channel activity. These devices position a singlecell in abutment with an aperture, so that the cell's membrane forms ahigh resistance, gigaohm seal, termed a gigaseal, around the aperture.The gigaseal allows channel currents across the cell membrane to bemeasured, by “whole cell” patch-clamp recording. Measurement of currentsin the presence and absence of potential modulators of channel activity,such as agonists and antagonists of receptors that couple with channels,provides a rapid and sensitive method for testing these modulators.Since changes in channel currents often are transient, the device alsofacilitates rapid perfusion of the cell with potential modulators andwash solutions. This allows rapid exposure and removal of themodulators. The device may be configured as a system that simultaneouslyand/or sequentially analyzes more than one single cell (see, amongothers, Example 12).

[0579] Embodiment 1

[0580]FIG. 51 shows a microfluidic device 1310 for measuring ioncurrents, in accordance with aspects of the invention. Device 1310includes a planar patch clamp electrode consisting generally of threelayers: a substrate layer 1312, a fluidic layer 1314, and a base layer1316.

[0581] Substrate layer 1312 includes one or more patchable orifices1318, of about 0.1-5 μm, or about 1-5 μm in diameter. The perimeter ofeach orifice forms a gigaseal with the membrane of a single cell beinganalyzed. Accordingly, substrate layer 1312 may be fabricated from anynonconducting material capable of forming a highly resistant seal, andmay be relatively hard. Suitable materials for the substrate layerinclude glass, silicon, and/or plastic, among others.

[0582] The substrate layer separates fluidic layer 1314 and base layer1316. The fluidic and base layers each are filled with one or morebuffer solutions that mimic the external and internal ionicenvironments, respectively, of single cells being analyzed. These buffersolutions may be referred to as external and internal buffers,respectively. The movement of ions through the cell membrane,effectively between the fluidic and base layers, creates currents thatcan be measured using sensitive amplification equipment. The fluidiclayer may be formed by any suitable technique, such as multilayer softlithography, for example, as described elsewhere in this DetailedDescription. The fluidic layer may be controlled by any suitable controlmechanism, such as an overlying microfluidic control layer 1320. Thebase layer may be formed out of any suitable material, such as glass,plastic, and/or an elastomeric material, among others. The base layermay be cut (punched), molded, etched, and/or embossed, among others, to(1) form a tight seal with substrate layer 1312, and (2) form areservoir holding internal buffer that is in fluidic contact with eachorifice and that accepts an electrode and/or electrode plate, typicallyconnected to suitable stimulation and recording equipment. In preferredembodiments, the bore of the patch clamp channel may be large enough topermit dislocation or dislodging of the particle from the patch clampwhen fluid flow is reversed through the bore of the patch clamp channel.

[0583] Embodiment 2

[0584] FIGS. 52-58 shows a microfluidic system 1340 for single-cellpatch-clamp recordings, in accordance with aspects of the invention.System 1340 includes a fluid-layer network 1342 and a fluid controllayer 1344, both formed by multilayer soft lithography, for example, asdescribed elsewhere in this Detailed Description. Network 1342 andcontrol layer 1344 position a single cell over a patchable orifice oraperture formed by a substrate layer (see below). Positioning the singlecell establishes an appropriate buffer gradient between fluid-layernetwork 1342 and a base-layer fluidic chamber, as described above forFIG. 51. Once a high-resistance seal is formed between the positionedcell and the substrate, around the orifice, system 1340 allows thepositioned cell to be perfused with one or more of a set of reagents,such as drugs, ligands (for the case of ligand-gated channels), bufferswith distinct ionic compositions, and/or wash solutions. Perfusion ofthese reagents permits rapid measurement of the effect of these reagentson the electrical activity of the cell.

[0585] To carry out these functions, system 1340 includes severalmechanisms that cooperate serially and/or in parallel. A cellmanipulation mechanism 1346 inputs, positions, and retains single cells.A cell perfusion mechanism 1348 exposes and washes the retained singlecells in a precisely controlled manner using a set of reagent-inputnetworks. An electrical monitoring mechanism 1350 electrically contactsboth the fluid-layer network 1342 and a base-layer fluidic chamber (notshown) to measure current, voltage, and/or resistance of retained singlecells before, during, and/or after exposure to desired reagents and/orelectrical manipulations.

[0586] Cell manipulation mechanism 1346 itself includes a set ofmechanisms, including a cell input mechanism 1352, a cell positioningmechanism 1354, and a cell retention mechanism 1356. These mechanismsact in a coordinated fashion to manipulate single cells for patch-clampexperiments.

[0587] Cell input mechanism 1352 generally comprises any mechanism thatacts through an input reservoir 1358 to introduce cells into fluid-layernetwork 1342. Input mechanism 1352 is similar to input mechanism 263 ofExample 2. Other suitable input mechanisms are described above, inSection IV.

[0588] Cell positioning mechanism 1354 generally comprises any mechanismthat acts to position single cells within microfluidic network 1342. Inaddition to simple flow channels, the cell-positioning mechanism mayinclude a focusing mechanism 1360. Focusing mechanism 1360 places inputcells in an input stream 1362 at a central portion of inlet channel1364, labeled “E1,” flanked by focusing flow streams from focusingreservoirs 1366, 1368, labeled “F1” and “F2.” Mechanism 1360 directsfluid from input and focusing reservoirs 1358, 1366, 1368 to junction1370 from three orthogonal directions. FIG. 53 shows an alternativecell-focusing mechanism 1372, in which cell-input and focusing streamsjoin at acute angles, forming an “arrowhead” configuration. Focusingmechanisms 1360 and 1372 are similar to aspects of positioning mechanism263 of Example 2.

[0589] Cell positioning mechanism 1354 stochastically segregates singlecells using a divided-flow mechanism 1374, downstream from focusingmechanism 1360 or 1372; see FIG. 54. Specifically, focused cells aredirected down inlet channel E1 and encounter a divided flow path 1376.Divided flow path 1376 directs fluid to a waste reservoir 1378 (seeFIGS. 52 and 53) through outlet channels 1380, 1382 (labeled “W1” and“W2,” respectively, in FIG. 54). These outlet channels include anarrowed portion 1384 and a size-restrictive channel 1386 that determinethe relative flow rate through each corresponding outlet channel.Narrowed portion 1384 has a substantially larger diameter thansize-selective channel 1386, so that most of the flowing fluid (andcells) passes through narrowed portion 1384. However, some fluid passesthrough size-restrictive channel 1386, eventually bringing a single cell1388 to the mouth of the channel.

[0590] Cell retention mechanism 1356 generally comprises any mechanismfor retaining a cell at a desired position, generally adjacent anorifice and/or electrode(s). Here, the cell retention mechanismfunctions at the channel mouth; see FIGS. 54 and 57. In particular, cell1388 cannot enter size-restrictive channel 1386 because the cell is toolarge. However, the pressure drop across size-restrictive channel 1386pulls cell 1388 against the channel mouth, holding cell 1388 inposition. Positioned cell 1388 may restrict or block flow throughsize-restrictive channel 1386, so that additional cells no longer areurged toward channel 1386. Cell 1388 also is positioned over an orifice1390 (see FIG. 56) defined by the substrate layer. In alternativeembodiments, single cells may be positioned and retained over an orificeby any suitable positioning and/or retention mechanisms, for example,those described elsewhere in this Detailed Description.

[0591] With cell 1388 in position over orifice 1390, flow from inputreservoir 1358 is terminated, but flow from focusing reservoir F1 and/orF2 continues. Continued flow from F1 and/or F2 may be used to preventadditional cells from stopping near cell 1388, which might interferewith measurements. In addition, continued flow from F1 and/or F2 ensuresthat buffer in the region surrounding cell 1388 is refreshed. To performwhole-cell recordings, reservoirs F1 and/or F2, and generally inputreservoir 1358, are filled with external buffer, so that all of fluidicnetwork 1342 is equilibrated with external buffer. In contrast,base-layer chamber, below orifice 1390, is filled with internal bufferfrom a lower face (or side) of the base layer, generally prior to cellinput. The contents of these reservoirs could be reversed, if the cellis positioned on the opposite side of the aperture, or for reasons ofexperimental design.

[0592] Positioned cell 1388 is pulled against orifice 1390 by applying avacuum from the base-layer chamber. This establishes a highly resistantseal, the formation of which can be measured as an increase inresistance between fluid-layer network 1342 and the base-layer chamber(below orifice 1390) using electrodes in each chamber. Generally,fluid-layer network 1342 serves as a ground, and a recording electrodeis positioned in the base-layer chamber. Once the seal is formed, theresulting patched cell can be measured for its baseline electricalactivity or properties.

[0593] After establishing this baseline, and/or using an average orcalculated baseline, the effect of reagents, such as drugs, may betested using perfusion mechanism 1348. FIG. 52 shows the general layoutof mechanism 1348, which includes a shield or wash reservoir 1394, and aseries of reagent reservoirs 1396, in this case five reservoirs, labeledD1-D5. Flow through inlet channels 1398 extending from reservoirs 1394,1396 is actively promoted by a pump 1400 in control layer 1344. Pump1400 acts in concert on all inlet channels 1398 to provide a uniformforce for delivering the reagents and wash buffer. In contrast, flowthrough each individual inlet channel 1398 is regulated by acorresponding control valve 1402 that determines whether fluid flowsthrough the inlet channel 1398. Valves 1402 are shown in more detail inFIGS. 53, 54, 56-58, where these valves are labeled V_(W), and V1-V5,corresponding to control of wash reservoir (“W”) and reagent reservoirsD1-D5, respectively.

[0594]FIG. 55 show perfusion mechanism 1348 in more detail. Perfusionmechanism 1348 controls exposure of cell 1388 to each selected reagentusing a regulatable fluid sheath or shield, similar to that describedfor perfusion mechanism 268 of Example 2. Wash reservoir W is filledwith external buffer, and the buffer is flowed past cell 1388 from washinlet-channel 1404 by opening valve V_(W). Specifically, focusing bufferfrom F1 and/or F2 entering chamber E1 pushes the wash buffer in alaminar flow pattern or sheath flow 1406 over cell 1388, against wall1408. Because wash inlet-channel 1404 is closer to cell 1388 than any ofthe reagent inlet channels 1398, sheath flow 1406 spaces and preventscontact of reagents flowed from any of the reagent inlet channels. Uponclosing valve V_(W), any flowing reagent rapidly contacts the cell, andrecordings can be made as desired. Accordingly, cell 1388 may be exposedrapidly to any reagents in reservoirs D1-D5 in a controlled manner byselective opening and closing valves V_(W) and V1-V5, allowingmeasurement of electrical responses in a correspondingly rapid timeframe. Therefore, ligands introduced through reservoirs D1-D5 may beused to study their antagonist or agonist activity on ligand gatedchannels, among others.

[0595] Microfluidic system 1340 may be configured in many suitable ways.For example, reagent inlet channels may unite, entering chamber E1through a common port, as shown in system 250 of Example 2 (see FIG. 8).In this way, each reagent is equally spaced by sheath flow 1406 of thewash buffer and thus will reach cell 1388 at the same time when thesheath flow is terminated. Furthermore, such a design would allowreagent mixing and dilution, as described above in Example 8.Alternatively, or in addition, a pump may be included to drive flow frominput reservoir 1358 and focusing reservoirs 1366, 1368. Furthermore,system 1340 may be modified to be reusable by including a cell removalmechanism, as described in Example 7. System 1340 may be modifiedadditionally or alternatively to include a parallel or serial array ofretention/analysis sites, for example, as described above in Examples3-5, or below in Example 12.

Example 12 Microfluidic System for Multiplexed Analysis of Cells byPatch Clamp

[0596] This example describes microfluidic systems for performingelectrophysiological analysis on one or more cells out of a set ofsingle cells; see FIGS. 59-61.

[0597] Background

[0598] Patch clamping is an electrophysiological method that relies onthe formation of a seal between a biological membrane (for instance, acell) and an aperture. This seal may facilitate the measurement of smallcurrents created by the passage of ions across the membrane. However,the seal generally should be tight, since current leakage around theseal may interfere with, or prevent, measurement of the small currentsacross the membrane.

[0599] The efficiency of seal formation is an important issue for thedevelopment of automated, high-throughput devices for screening drugsbased on electrophysiological effects on cells. In manual patch-clampsystems, the efficiency with which cells can be successfully analyzedvaries, but very skilled technicians typically achieve properly sealedpatches at an efficiency of only about 50%. A similar efficiencyachieved by an automated device would require the device to“cherry-pick” wells containing properly sealed patches for use in drugscreens, limiting the utility of such a device. Furthermore, even whenproperly sealed patches are formed, more than one cell may need to beanalyzed to identify a typical or average cell response. Thus, there isa need for an automated device that more efficiently forms sealedpatches on cells, facilitating averaged analysis of multiple cells andreducing problems associated with cell-to-cell variation inelectrophysiological response.

[0600] Description

[0601] This example provides a multiplexed version of a single-aperturemicrofluidic device, with a defined number (“n”) of individuallycontrollable apertures. Each individually controllable aperture may beused to analyze a single cell by patch-clamp methods. Because only onepatched cell is required to form an effective seal for each experiment,the use of multiple apertures increases the probability of forming thisseal with the device. In addition, the device allows each aperture, andits associated cell, to be included in, or excluded from, an analysis.Thus, signals may be obtained from each individual cell that issuccessfully sealed by electrically isolating each correspondingaperture. Alternatively, or in addition, an “averaged” signal may beobtained from two or more of the individually controllable apertures,either by averaging separate measurements or measuring from two or moreapertures concurrently. Averaged signals may improve the robustness ofany data obtained.

[0602] Single-aperture Embodiment

[0603]FIG. 59 shows a one-aperture device 1430 to illustrate how each ofthe n apertures is structured. Device 1430 directs a single cell 1432into abutment with an aperture 1434. Aperture 1434 connects chambers1436, 1438. These internal and external chambers, 1436 and 1438,respectively, carry buffers whose compositions resemble that of theinternal (cytoplasm) and external (extracellular) environments,respectively, of cell 1432. A vacuum may be applied to internal chamber1436 to pull cell 1432 toward aperture 1434, forming a seal between thecell and aperture. Sealing and rupture of the cell membrane (whole cellentry) make the inside of cell 1432 electrically continuous withinternal chamber 1436. In other embodiments, the membrane may be leftunruptured but perforated, for example, by addition of channel-formingagents to internal chamber 1436, or the membrane may be left unrupturedand unperforated.

[0604] Electrical measurements then may be obtained. External chamber1438 may be connected to ground, while internal chamber 1436 may carry arecording electrode, generally connected to an amplifier. Ions passingthrough the membrane of cell 1432 create a current that may be measuredfollowing amplification with the amplifier. Device 1430 may be used tomeasure changes in ion channel-associated and/or transporter-associatedcurrents in the presence of potential drug candidates or othermodulators.

[0605] Multi-Aperture Embodiment

[0606]FIG. 60 shows a microfluidic device 1450 that is a multiplexedversion of device 1430, in accordance with aspects of the invention.Device 1450 may include a shared internal chamber 1452 that extendsaround the perimeter of device 1450. Internal chamber 1452 may connectto a shared external chamber 1454 using a plurality of apertures 1456,in this case, four. Each aperture may be isolatable, both electricallyand fluidically, using control valves 1458 (V_(N), V_(S), V_(E), andV_(W)). In addition, each aperture may be disposed immediately adjacenta cell retention mechanism, such as retention site or trap 1460. Traps1460 may be arranged so as to facilitate parallel loading from a singlesuspension of cells (one reservoir) or from plural suspensions of cells(plural reservoirs). Internal chamber 1452 may be connected to a vacuumsupply, and a recording electrode and ground may be connected toexternal and internal chambers, 1452 and 1454, respectively.

[0607] Device 1450 may be readied and used as follows. First, internalchamber 1452 may be loaded with internal buffer at internal-chamber port1462 (Port I), so that internal buffer is loaded up to apertures 1456.Next, open valves V_(N), V_(S), V_(E), and V_(W) may be closed, andcells may be loaded as a suspension using an input mechanism at a commoninput port 1464 (Port C). Then, the cell suspension may flow from Port Cto output reservoirs 1466 (“outlet”). Single cells may be positioned andretained at each trap 1460 (N, S, E, W) using any suitable positioningand retention mechanisms, such as those described elsewhere in thisDetailed Description, for example, Examples 1-3. Once a desired numberof cells are retained by retention mechanisms, device 1450 may be usedfor cell analysis. The vacuum supply may be turned on, and one or morevalves at a time may be opened to form an electrical connection betweenthe internal and external chambers, through the corresponding aperture1456. The resistance of the connection may be used to determine if asufficient seal has been produced at the aperture, with the membrane ofthe retained cell. If so, recording may be commenced.

[0608] Device 1450 may be modified in any suitable fashion,incorporating any suitable microfluidic mechanisms, such as thosedescribed in this Detailed Description. For example, device 1450 may bestructured to load cells serially and/or in parallel, as described abovein Examples 3-5. Furthermore, device 1450 may be included in an array ofsuch devices to form a microfluidic array. Alternatively, or inaddition, device 1450 may include a perfusion mechanism, such as thatdescribed in Examples 2 and 8, to allow precise delivery of selectedreagents, to individual cells or to a plurality of cells, serially or inparallel. Similarly, device 1450 may measure electrical parameters ofcells serially, that is, by using one aperture at a time, or inparallel, by using two or more apertures at a time, to obtain a summedreading of all connected apertures.

[0609]FIG. 61 shows data from a simple statistical analysis illustratinga few of the advantage of a multiplexed patch-clamp system, such assystem 1450. The fractional probability of successfully obtaining a sealin a well containing n apertures, P_(n), is related to the fractionalprobability of failed seal formation, P_(f), at a single aperture by theequation P_(n)=1−P_(f) ^(n). The probability of successful sealformation for a single aperture, P_(s), is related to P_(f) by theequation P_(f)+P_(s)=1. Therefore, if a seal is obtained successfully in50% of attempts, then with 4 apertures, P₄=1−(0.5)⁴=1−0.0625=0.9375.This corresponds to a 93.75% chance of obtaining at least one seal amongthe four apertures. FIG. 61 graphs the relationship between n (x-axis)and P_(s) (y-axis), with curve 1474 indicating (n, P_(s)) pairs thatgive a 95% probability of at least one of the n apertures forming asuccessful seal. (Apertures are called “channels” in FIG. 61.) P_(n)approaches unity, as P_(s) and/or n are increased.

Example 13 Multilayer Mold-Fabrication Method of Varying Height and/orCross-Sectional Geometries of Molded Microfluidic Structures

[0610] This example describes a method for producing, by softlithography, microfluidic devices in which the cross-sectional geometryand/or height of structures within and/or between microfluidic networksvary; see FIGS. 62-71.

[0611] Background

[0612] A microfluidic network may include structures having a variety offunctions. For example, regulatable channels may include deflectablevalves, acting to partially or completely close the channels and/or topropel fluid through the channels. These channels generally are formedwith a semicircular or arcuate cross-sectional geometry to enableefficient valve closure. By contrast, particle-positioning channels mayact primarily as conduits for particles carried by fluid. Theseparticle-positioning channels generally have a height sufficient toallow particle movement. Accordingly, particle-positioning channels maybenefit from a rectangular cross section to enable particles to moveunrestrictedly from side-to-side (transversely) within the channels.Such unrestricted movement may allow particles to occupy a greaterproportion of the width of the channels, rather than just the centralportion, as with arcuate channels. Other channels may be size-selectiveor particle-restrictive, preventing entry of particles greater than agiven size. These particle-restrictive channels may have a height thatis less than the diameter of particles of interest. Furthermore,microfluidic networks may include cell/culture chambers with roofheights that are greater than more narrow channels, as described inExample 10, to improve the functionality of the chambers. Therefore,these and other structures described elsewhere in this DetailedDescription may benefit from, or require, roof height to vary in orderto function properly.

[0613] Single-layer molds often are formed using a desired thickness ofphotoresist on a substrate. The photoresist is patterned using acorresponding template that allows selective light exposure andphotosensitization of patterned regions of the photoresist. Depending onwhether the photoresist is positive or negative, the selectively exposedregions are either resistant or sensitive, respectively, to subsequentremoval during development with a suitable developing agent. Thisdevelopment nonspecifically removes all sensitive regions, generallydown to the substrate. The resistant regions are generally rectangularin cross-section, but may be heated to round their edges into anrounded/arcuate configuration. Accordingly, these remaining regions ofthe resulting mold may produce microfluidic channels of complementarystructure using soft lithography. In other embodiments, multiple layersof photoresist may be built up by sequential coating, masking, and

[0614] Despite the importance of varying height and/or cross-sectionalshape across a microfluidic network, molds formed from a single layer ofselectively removable material, such as photoresist, may not allowsufficient flexibility in the structure of a microfluidic network formedfrom the mold. For example, the depth to which the single layer may beremoved cannot be varied readily, producing features of a single height,generally equal to the thickness of the single layer. Similarly,cross-sectional geometry may be difficult to vary within a single layerof the mold. Treatments that alter cross-sectional geometry, such asheating, also may act nonselectively across the single layer. Therefore,a method is needed for forming a mold using plural selectively removablelayers.

[0615] Description of Method

[0616] The method described in this example may be used to form channelswith different cross-sectional geometries and/or heights at distinctpositions within a microfluidic network. A mold is fabricated usingplural layers of photoresist that are each individually patterned,selectively removed according to the pattern, and optionally rounded byheating. Thus, each of the plural layers may contribute only a subset ofthe resulting mold, so that the mold's relief pattern is the sum of theremaining portions from each of the plural layers. Using the mold toform a microfluidic network allows various types of channels or otherpassages to be formed. Channels with a rounded/arcuate cross-sectionalshape may be formed in sections of the network where valves are needed.These sections may be connected with other portions of the network thatare formed to have a rectangular profile, to promote particle movementand to enable precise delivery of one or more particles to a specificarea of a microfluidic network. The specific area can be as small as thedimension of a single particle, such as a cell. These structures andother suitable microfluidic structures may be produced using the methoddescribed below. This method focuses on formation of a fluid layer, butmay be suitable for any portion(s) of a microfluidic system, including acontrol layer or a base layer (see Example 11).

[0617] A fluid-layer mold is fabricated in a first series of steps bymicromachining techniques. The fluid-layer mold may be used subsequentlyin a second series of steps, as described below, to mold a complementarymicrofluidic layer by soft lithography. FIGS. 62-68 illustrate howfluid-layer mold 1480 may be formed by sequentially disposing,patterning, and selectively removing three layers of photoresist on orabove a silicon wafer. Each layer is formed at a desired thickness byapplying the photoresist, and then rotating the wafer according to adefined rotational profile to produce the structure of FIGS. 62, 64, and67. Next, the photoresist is baked, patterned by exposure to UV light,and then developed to selectively remove portions of each layer, shownin FIGS. 63, 65, and 68. To mold closable channels, a photoresist layermay be baked at high temperature to round remaining portions, shown inFIG. 66. Each individual step is detailed further below.

[0618] The first layer may be applied directly to a bare silicon wafer(the substrate). The first layer may have any suitable thickness, inthis case 5 μm, and may be formed with any suitable material, such as anegative photoresist, SU8 2005 (Microchem, Newton, Mass.). Afterapplication of the negative photoresist, the wafer may be rotatedaccording to a suitable rotational protocol to achieve a desiredthickness and consistency. For example, the wafer may be rotated asfollows: rotate to 500 rpm over 5 sec, maintain at 500 rpm for 5 sec,ramp to 3000 rpm over 8 sec, and then maintain at this speed for 30 sec.Then the rotation may be halted and the wafer heated according to asuitable heating protocol. For example, the wafer may be heated for 1min at 65° C., 2 min at 95° C., and finally 30 sec at 65° C. Thisheating process may drive off the solvent in which the photoresist maybe supplied. FIG. 62 shows mold 1480 with substrate 1482 carrying firstlayer 1484. The relative sizes of components here and in related FIGS.63-69 are not drawn to scale.

[0619] The first layer may be patterned and selectively removed asfollows. A desired template may be positioned in contact with the firstlayer and then exposed to UV light, 160 J/cm². Next, the substrate/firstlayer may be subjected to a suitable post-exposure heating protocol,such as: 1 min at 65° C., 2 min 30 sec at 95° C., and 30 sec at 65° C.Unpolymerized (unexposed) first layer may be washed away with anysuitable developer, such as that supplied by Microchem, followed bywashing with acetone and then isopropanol. Then, the first layer may besubjected to a suitable post-development heating protocol, such as 1 minat 65° C., 5 min at 95° C., and then 30 sec at 65° C. This heatingprotocol may be followed by a post-development exposure with UV light,400 J/cm². FIG. 63 shows mold 1480 with first layer 1484 contributingfirst-layer relief-structure 1486 (residual first layer), which may havea height of 5 μm.

[0620] The second layer may be added next and may have any suitablethickness, in this case a thickness of 20 μm formed by spin coating.First, mold 1480 may be treated with hexamethyldisilazane (HMDS) for 10min. Next, a suitable patternable material, such as a positivephotoresist, PLP 100 (AZ Electronic Materials/Clariant Corporation) maybe applied. Application may be by spin coating, using any suitableprotocol, such as the following: spin the wafer at 500 rpm, dispense thepositive photoresist to the wafer/residual first layer over 14 sec, spin15 sec, ramp to 2000 rpm over 5 sec, and maintain at this speed for 30sec. Rotation then may be stopped, and the second layer may be baked for2 min at 100° C. FIG. 64 shows mold 1480, at this intermediate stage,carrying second layer 1488, which covers first-layer relief-structure1486.

[0621] The second layer may be patterned and selectively removed asfollows. Any suitable template may be positioned in contact with thesecond layer and exposed to UV light, 450 J/cm². Next, the second layermay be developed (selectively removed) by any suitable protocol, such as3 min. in AZ 400K 1/3 with deionized water. FIG. 65 shows mold 1480after patterned removal of both first and second layers 1484, 1488.First-layer relief-structure 1486 and a second-layer relief-structure1490 may have distinct heights based on the thickness of photoresistfrom which they are formed.

[0622] Second-layer relief-structure 1490 may be rounded by any suitableheating protocol. For example structure 1490 may be rounded by thefollowing heating protocol: ramp from 70° C. to 100° C. (1° C./min),maintain 60 min at 100° C., ramp to 200° C. (1° C./min), maintain 60 minat 200° C., and ramp down to 40° C. (1° C./min). FIG. 66 shows how thisheating protocol may convert rectangular second-layer relief-structure1490 (FIG. 65) to rounded second-layer relief-structure 1492.

[0623] A third layer may be added next and may have any suitablethickness, for example, a thickness of 20 μm. A suitable selectivelyremovable material, such as negative photoresist SU8 2050 (Microchem),may be applied to the wafer carrying the residual first and secondlayers. Spin coating may be achieved by the following protocol: thewafer is ramped to 500 rpm over 5 sec, maintained at this speed for 5sec, ramped to 5000 rpm over 17 sec, and maintained at this higher speedfor 30 sec. The rotation is stopped. Next, the third layer may be heatedby any suitable, such as: 2 min. at 65° C., 3 min. at 95° C., and 30 secat 65° C. FIG. 67 shows third layer 1494, which covers first-layer andsecond-layer relief-structures 1486, 1492 at this stage.

[0624] The third layer may be patterned and selectively removed asfollows. A desired template may be positioned in contact with the thirdlayer and exposed to UV light, 310 J/cm². The exposed layer may beheated by any suitable protocol, such as 1 min. at 65° C., 4 min. at 95°C., and 30 sec at 65° C. Next, the third layer may be selectivelyremoved with a suitable developer, such as that of Microchem, and thenmay be washed with acetone followed by isopropanol. Subsequently, thethird layer may be subjected to a suitable post-development heatingprotocol, such as 1 min. at 65° C., 5 min. at 95° C., and 30 sec at 65°C. Finally, the third layer may be exposed to UV light in apost-development exposure of 500 J/cm². FIG. 68 shows mold 1480 having athird-layer relief-structure 1496.

[0625] Any suitable aspects of the method described above may bemodified, and any patternable, selectively removable material may beused. In addition, any suitable number of layers may be used.Furthermore, each layer may have any desired thickness, according to theheight of a desired relief structure. When optically patternable layersare used, each layer may be negative or positive photoresist, and may beused to form a rectangular or rounded cross-sectional profile. Reliefstructures formed by distinct layers may be nonoverlapping, partiallyoverlapping, and/or completely overlapping in specific regions or allregions of the mold. Accordingly, relief structures may represent thesum of plural selectively removed layers.

[0626] An exemplary method for forming a control-layer mold is asfollows. The mold may be fabricated from a single layer of positivephotoresist. A 20-μm layer of suitable photoresist, such as positivephotoresist PLP 100, may be applied, patterned, selectively removed, androunded as described above for the second layer of the fluid-layer mold.

[0627] The fluid-layer and control-layer molds fabricated above may beused to mold a microfluidic chip using any suitable material,particularly an elastomeric material, such as polydimethylsiloxane(PDMS). Exemplary PDMS elastomers are General Electric Silicones RTV615, produced from a two-component mixture of a prepolymer/catalyst anda crosslinker. In this two-component mixture, the prepolymer/catalyst(component A) is a polydimethylsiloxane bearing vinyl groups and aplatinum catalyst, and the crosslinker (component B) bears siliconhydride (Si—H) groups. Using these specific components, components A andB may function optimally at a ratio of about 10:1 (A:B). However,“offratios” above and below this ratio may be used for the fluid-layermembrane and the control layer to promote subsequent bonding. Forexample, the control layer may be formed at a ratio of about 4:1, toprovide rigidity and thus mechanical stability, and the fluid-layermembrane at a ratio of about 30:1. The excess of either component A or Bin these two layers remain reactive near the membrane surface.Accordingly, these two layers may be abutted and bonded by post-curingwith baking to fuse these layers into a monolithic structure (seebelow).

[0628] The fluid-layer and control-layer molds may be fabricated andjoined as follows. After treatment with trichloromethylsilane (TCMS), arelatively thin PDMS membrane, for example, about 50-150 μm, may be spunon completed fluid-layer mold 1480. FIG. 69 shows a membrane 1498 beingformed on fluid-layer mold 1480. In addition, a thicker PDMS layer, forexample, approximately 5-10 mm, may be formed on the control-layer mold.After suitable first-step curing, such as 90 min at 80° C., the controllayer may be detached from the mold, cut, and punched to interfaceproperly with control lines of the control layer. Then, this controllayer may be aligned with the fluid layer, while the fluid-layermembrane 1498 is still attached to the fluid-layer mold. Once assembled,the fluid and control layers may be cured a second time to chemicallybond them, using a post-curing step of heating for about 3 hours at 80°C. After post-curing, the resulting chip may be detached from thefluid-layer mold, cut, and punched to create fluid reservoirs thatinterface at desired positions with channels. Finally, the chip may bebonded to a suitable substrate, such as a glass cover slip, to completethe fluid channels.

[0629] The post-curing step may be modified to enhance compatibilitywith cells. Lower ratios of PDMS components A and B, such as 4:1 (A:B),tend to be toxic to cells, particularly during cell culture. Thistoxicity may be due to a diffusible, toxic material(s) in the controllayer. Thus, when a much thicker control layer, formed at a ratio of4:1, is fused to a thin fluid-layer membrane, formed at a ratio of 30:1,the resulting monolithic structure may have the toxic characteristics ofa 4:1 layer, even within the fluid-layer portion. However, suitabletreatment of the control layer, either alone in contact with the fluidlayer membrane, reduces or eliminates this toxic characteristic.Suitable treatments that remove or modify the toxic material may includeexposure to heat, a chemical (such as a gas, a liquid, a plasma, etc.),radiation, light, and/or the like. (Such treatments also may reduce themovement of fluids within the channel, or components thereof, into thechip.) In some embodiments, longer post30 curing at elevated temperaturemay remove or modify the toxic material(s), enhancing the effectivenessof the resulting chips for cell experiments. Such a longer post-curingstep may be conducted for about 6 hours, 12 hours, or more preferablyabout 24 hours or more at about 80° C.

[0630] Images of Molds and Chips

[0631]FIGS. 70 and 71 show photographic images of fluid-layer molds andthe corresponding microfluidic chips formed with these molds. Themicrofluidic networks represented here, have been shown and described insystem 1340 of Example 11 (FIG. 70) and in a modified form in system 850of Example 7 (FIG. 71). Distinct regions of each mold and fluid layerare indicated by letters A, B, and C. Area A corresponds to roundedsecond-layer relief-structures 1492 described above. These areas arecolor-coded in blue on many of the figures presented above. Channels ofarea A are about 200 μm wide and approximately 20 μm high. Area A may beused to form valves and pumps by overlapping control lines from acontrol layer with this area, such as valve 1500 in FIG. 71. Area Bcorresponds to third-layer relief-structure 1496. These areas arecolor-coded in red on many of the figures presented above. Channels ofarea B have a rectangular profile, approximately 100 μm wide and 20 μmhigh. These channels enable precise particle control, because they allowparticles to distribute across the width of the channel, following thewalls and/or the center of a fluid stream(s). Such channels may be usedto drive particles to precise areas of each chip. Area C corresponds tofirst-layer relief structure 1486. These areas are color-coded inturquoise on several of the figures presented above. These channels havea rectangular profile, 10 μm wide and 5 μm high. Small channels of thistype are used in combination with channels of area A or B to trap cellsor beads. Fluid may flow in these channels entry of cells or beads maybe restricted.

Example 14 Detection System for Kinetic Analyses in Microfluidic Systems

[0632] This example describes a detection system, including amodulation-demodulation method and the use of tracer materials, foranalysis of kinetic reactions involving particles in microfluidicsystems; see FIGS. 71A-F.

[0633] Background

[0634] Microfluidic systems may be used to measure the kinetics of manyaspects of cellular metabolism. However, metabolic processes ofphysiological significance can occur at substantially different rates,with characteristic times that may range from microseconds (10⁻⁶ sec) orless to days (10⁵ sec) or more. Therefore, detection methods are neededto measure cellular events that occur at these vastly differing rates.

[0635] Time-resolved fluorescence spectroscopy has been one of the mostpopular approaches to cellular kinetics studies. Typically, dyemolecules are introduced into cells, and emission from the molecules isproduced by excitation with an intense light source (such as an arc lampor laser). The intensity of this emission is monitored over the courseof the analysis to infer the kinetics of a process under study. However,the emission intensity of the dye molecules may be reduced orextinguished over time by photobleaching. As a result, some cellularprocesses that occur over relatively longer time periods may be moredifficult to monitor in a microfluidic system due to thisphotobleaching.

[0636] Because the rate of photobleaching is related to the intensity ofexciting light, a weaker light source may be used to reduce this rate.For example, FIG. 71A shows a comparison of photobleaching rates versustime using a relatively stronger laser (1.6 mW) and a relatively weakerlaser (1.6 μW). However, the exciting light source produces a reducedemission signal and signal-to-noise ratio, since the emission signal isproportional to the illumination intensity. Therefore, microfluidicanalyses would benefit from a detection system that reducesphotobleaching, increases the ratio of signal-to-noise, and/or allowskinetic analysis of both fast and slow processes.

[0637] Description of Detection System

[0638] This example describes an exemplary detection system for use withmicrofluidic assays, in accordance with aspects of the invention. Thedetection system may include a modulation-demodulation mechanism; seeFIGS. 71B-71E. This mechanism may improve signal-to-noise ratios,allowing use of weaker light sources, and/or reduce photobleaching,allowing use of stronger light sources. The detection system also mayinclude a method using tracer dyes to measure initiation of rapidkinetic reactions with particles; see FIG. 71F.

[0639] Light Detection Device

[0640]FIG. 71B shows an exemplary system 2010 for detecting an opticalsignal from a sample. System 2010 may include a light source 2012,optics 2014, a detector 2016, a digital storage device 2018, and amodulation-demodulation mechanism 2020.

[0641] Light source 2012 may be used to illuminate one or more particleswith light to visualize the particle and/or to perform an assay. Thelight source may generally may include any mechanism for producing lighthaving the desired characteristics, including time-dependent and/orcontinuous light sources. Suitable examples may include a laser, alight-emitting diode (LED), or a lamp, among others.

[0642] Optics 2014 may be used to receive light from light source 2012and direct the light at the particles and/or to receive light from theparticles and direct it to detector 2016. Optics may mediate anysuitable alteration of light to facilitate analysis, includingrefraction, reflection, diffraction, polarization, attenuation, spectralalteration, and/or scattering, among others. Suitable optics may includelenses, mirrors, fiber optics, filters, gratings, etalons, and/or thelike. Exemplary optics may include a conventional microscope or othersuitable optical device that is separate from, or partially or whollyintegrated with, a microfluidic system.

[0643] Modulation-demodulation mechanism 2020 may include a modulator2022 and/or a demodulator 2024. Modulator 2022 generally comprises anymechanism to provide time-dependent variation in the intensity ofexposure of sample to source 2012. This variation may be intrinsicand/or extrinsic to the light source. Intrinsic modulation occurs whenthe light source itself changes in intensity, as with a pulsed or strobelaser (such as a diode laser). Such a pulsed laser may be pulsed veryrapidly, up to millions of pulses per second, allowing forhigh-frequency illumination of particles. Extrinsic modulation occurswhen the light source is continuous (or quasi-continuous), but adownstream mechanism alters the intensity of light before it is incidenton the sample. Suitable extrinsic modulators include optical chopperwheels, Pockels cells, Kerr cells, acousto-optic modulators, and/orelectro-acoustic and other modulation devices. By contrast, demodulatorsgenerally comprise any mechanism for interpreting signals from detector2016 based on the activity of the modulator. The control and interplaybetween the modulator and demodulator may be performed using anysuitable mechanism, such as lock-in amplification using custom-designedand/or commercial devices.

[0644] Detector 2016 may be used to detect light, rapidly and/orrepeatedly, and convert the detected light into representativeelectrical signals. Such a detector may include a photomultiplier tube,avalanche photodiode, and/or other photodetector that provides theability to rapidly detect light signals produced by a source 2012illuminating the particles. Collecting light emitted through opticalfilters into photomultiplier tubes or other photodetectors may enableconversion of photons to electrons for collection of quantitativeinformation.

[0645] Digital storage device 2018 may digitize and/or store electricalsignals received from detector 2016. These stored signals may beretrieved, corrected, and/or otherwise converted or manipulated, andprinted or displayed, as desired.

[0646] Exemplary Results using a Modulation-Demodulation Mechanism forMicrofluidic Analysis

[0647]FIG. 71C shows a comparison of signal-to-noise ratios over timewithout (top) and with (bottom) source and signalmodulation-demodulation. In this example, an embodiment ofmodulation-demodulation mechanism 2020 boosts the signal-to-noise ratioby a factor of over 2000-fold. Accordingly, weaker light sources may beused and an emitted fluorescence signal may be measured over a longertime course.

[0648]FIG. 71D shows use of an embodiment of mechanism 2020 to determinethe rate at which a reagent-particle interaction occurs in a singleexperiment. Here, a biotinylated bead has been loaded into a trap on amicrofluidic chip, such as a chip designed according to system 250 ofExample 2. Dye-labeled streptavidin (reagent) is exposed to the bead ina pulsatile fashion, using cycles of staining and washing controlled byautomated operation of control valves. In this case, each ten-secondcycle includes a two-second exposure to reagent, followed by aneight-second exposure to wash buffer. Each cycle produces a spike influorescence intensity. However, the average fluorescence intensityachieves a near-maximal level in about twenty cycles. Accordingly,maximal staining occurred in about forty seconds (twenty cycles timestwo seconds per cycle). Therefore, flow-based exposure and washing maybe optimized to avoid time- and labor-intensive labeling and washingsteps, and to minimize use of reagent. The pulsatile exposureillustrated here may be used with any suitable particle and dyecombination to measure the rate at which interaction occurs.

[0649]FIG. 71E shows the ability of an embodiment of the microfluidicdetection system to measure a kinetic response of signal transduction ina cell. A calcium sensor dye, Fluo-3, was loaded into a cell, and thecell was trapped in a microfluidic chip, such as a chip designedaccording to system 250 of Example 2. The trapped cell was stimulatedwith ionomycin, at about time=120 sec, to promote release ofintracellular calcium. The graph shows intensity of fluorescence,corresponding to intracellular calcium concentrations, versus time. Suchan analysis measures the response of an individual cell, so compensatoryoscillations in calcium levels are visible.

[0650] Method Using Tracer Dyes

[0651] Most rapid reactions or events are difficult or impossible tomeasure unless their starting points can be precisely defined.Accordingly, a tracer material, such as a tracer dye, may be included ina reagent of interest to indicate the time at which fluid containing thetracer dye and reagent contacts a particle(s). Thus, first detection ofthe tracer dye in contact with the particle defines a zero time point atwhich a reaction or event was initiated.

[0652] The tracer dye may have any optically detectable property and maybe inert or reactive. Suitable optically detectable properties aredescribed above in Section VIII. Inert dyes generally do not contributedirectly to a detected assay result. Therefore, inert dyes generally donot affect cellular metabolism, and may not interfere optically orchemically with reagent dyes used to measure information aboutparticles. Inert dyes may be nonbinding or binding. Nonbinding dyes donot bind to particles and may simply mark fluid volumes. Binding dyesmay bind to particles, but do not contribute directly to a detectedresult from particles. By contrast, reactive dyes react with particlesand contribute to a detected result. Suitable reactive dyes may bedetectable when first combined with particles, but may show a change inan optical property during an assay. Inert or reactive dyes may beexcluded from cells, may partition into particles, or may be transportedinto the interior of cells. Inert and reactive dyes that may be suitableare sold by Molecular Probes, Eugene, Oreg.

[0653] Rapid perfusion mechanisms, such as perfusion mechanism 268 ofExample 2 above, coupled with a tracer dye and detection systemdescribed in this example, may allow very rapid analyses to be performedon particles. Such rapid analyses may measure events that occur in lessthan about 2 sec, 1 sec, or 500 msec. Furthermore, these rapid analysesmay be performed on living cells to measure cell responses that are notdetectable readily by other methods.

[0654]FIG. 71F shows use of an embodiment of modulation-demodulationmechanism 2020 and a tracer dye in a microfluidic system to measure therate at which reagent is exposed to particles. A perfusion mechanism,such as mechanism 268, was used to expose a retention site to afluorescent dye. The resulting increase in fluorescence was measuredover time. At time “T,” an electrical signal was sent to a valvecontroller. After a short mechanical delay of about 5 msec, fluorescencemeasured at the retention site begins to increase, reaching a maximumvalue in less than 100 milliseconds. Accordingly, rapid kinetic analyseson a millisecond time scale may be performed using microfluidic systemsdescribed herein.

Example 15 Microfluidic Analysis of a Heterogeneous ParticlePopulation—Part I

[0655] This example describes microfluidic systems for sorting andanalyzing heterogeneous populations of particles, particularly cells,based on differences in particle size; see FIG. 72.

BACKGROUND

[0656] Heterogeneous cell populations, such as blood, present achallenge for rapid analysis. Cells of interest in blood generally needto be separated from other cells that are of less interest to avoidinterference from these other cells. Accordingly, blood may need to betreated/manipulated to selectively lyse, coagulate, pellet, bind, and/ormodify, among others, specific cells within the blood. Suchmanipulations add to the time and expense required for analysis ofblood, because they involve trained personnel, expensive equipment,lengthy incubations, repeated transfer of relatively large volumes ofreagent or sample, and/or the like. In addition, such manipulationsexpose personnel to increased risk of exposure to infectious agents inthe blood. As a result, many diagnostic procedures using whole blood areexpensive and slow. Therefore, integrated systems are needed thatautomatically sort and analyze heterogeneous cell populations on amicrofluidic scale.

[0657] Description

[0658] This example describes microfluidic systems that sorts bloodcells and other heterogeneous particle populations according todiameters of individual particles. With these systems very small volumesof blood may be sufficient for statistically significant diagnoses orprognoses. Such systems may facilitate analysis of patient samples withimproved speed, accuracy, safety, and/or cost, among others.

[0659]FIG. 72 shows a microfluidic system 1520 sorting cells. System1520 is based on system 250 of Example 2 and includes positioning andretention mechanisms 264, 266 described in that example. A blood samplewas introduced into system 1520 and directed toward retention chamber270. Cells 1522 of this sample include red blood cells and platelets,but do not include detectable white blood cells, which would be retainedby the retention mechanism due to their larger diameters. Cells 1522enter chamber 270 but exit through size-selective side-wall channels300. FIGS. 72A-D show time-lapse video images that include cells inchamber 270 and in channels 300. White blood cells such as lymphocytes,monocytes, and granulocytes (neutrophils, eosinophils, and basophils),when present, would be retained in chamber 270. These white blood cellsare too large to pass through channels 300. Therefore, system 1520 maybe used to separate red blood cells and platelets from white bloodcells, for selective analysis of the white blood cells (or red bloodcells) in the system.

[0660] System 1520 may be modified to select plural populations ofparticles of different size. For example, the system may be modified toinclude a serial set of retention mechanisms. Outflow throughsize-selective channels 300 for each retention mechanism 270 may bedirected partially or completely toward an input site of a successiveretention mechanism. Each successive mechanism may have a reduceddiameter of channel 300, so that a reduced diameter of particle isretained in each successive mechanism. With this arrangement, largerparticles are retained earlier in the series of mechanisms, whereassmaller particles are retained later in the series. Any suitableretention mechanism may be used at each position in the series.

[0661] Particles retained in the retention mechanism of system 1520 orrelated systems may be treated and analyzed. Particles may be treated byexposing them to desired reagents, for example, using perfusionmechanism 268 of Example 2, or by introducing reagents from any otherreservoirs included in system 1520. Thus, particles retained in distinctretention mechanisms may be isolated and exposed to distinct reagents,as described in Example 4. Systems such as system 1520 may enableon-chip staining and washing, eliminating any need for multiple pipetingand/or centrifugation steps during manipulation and detection.

[0662] Suitable characteristics of retained particles may be detected byflow or scanning cytometry, among others. In flow cytometry, particlesare detected while flowing past a detection mechanism, such as a lightsource coupled to a photodetector. Accordingly, particles may bereleased from each retention mechanism, for example, using a releasemechanism, such as described above in Example 7, to flow past adetector. Alternatively, or in addition, characteristics of particlesmay be detected or otherwise detected while the particles are relativelystationary, such as when localized in chamber 270. Photons may beconverted to electrons using photomultiplier tubes, avalanchephotodiodes, CCDs, or similar technologies. Light emitted from dyes maybe bright enough to detect using a single CCD, and scattered light mayyield enough structural information from particles, when combined withfunctional information, to identify specifically the type and state ofparticles.

[0663] Additional aspects of sorting a heterogeneous particle populationare described below in Example 26.

Example 16 Microfluidic Interaction of Specific Binding Pairs on Beads

[0664] This example describes detection of interaction between aspecific binding pair, biotin and avidin, on beads in a microfluidicsystem; see FIGS. 73-74.

[0665] Background

[0666] Beads are used frequently by pharmaceutical and biotechnologycompanies as carriers for drug targets, drug candidates, chemicalsyntheses, immunoassays, chromatography, and/or so on. However, smallnumbers of beads are difficult to manipulate, particularly to detectreactions that occur rapidly. As a result, using currently availabletechnology, assays with beads generally are conducted on a relativelylarge scale, wasting valuable reagents and/or may measuring a reactionendpoint that misses valuable earlier reaction information. Therefore,systems are needed to study interaction, including rapid interactions,using small numbers of beads.

[0667] A specific binding pair, biotin/streptavidin, was selected forinteraction on beads; see FIG. 73. Biotin is a vitamin with a molecularweight of 244 daltons. Its partner, avidin, binds biotin with fiercetenacity, being the strongest non-covalent attachment known, with anassociation constant of 10¹⁵ M⁻¹. This binding reaction has been studiedintensively for many decades, and there is a rich literature. The greatstrength of this binding suggests that it might be a good model systemfor the study of biological binding reactions in general. It has alsoformed the basis for many detection and signal amplification strategiesfor both research and clinical labs.

[0668] Avidin and streptavidin are vertebrate and bacterial biotinpartners, respectively. Avidin is a protein with a molecular weight ofabout 68 kilodaltons, including four identical subunit chains, each 128amino acids long. Avidin is found predominantly in the egg white ofbirds, amphibia, and reptiles. The protein streptavidin, produced by thebacterium Streptomyces avidinii, has a structure very similar to avidin,also binding biotin tightly. However, streptavidin often exhibits lowernonspecific binding, and thus is frequently used in place of avidin.

[0669] Method

[0670] Materials for measuring biotin/avidin interaction were asfollows. A microfluidic chip was fabricated based on system 250 ofExample 2. Beads, 6.7-micron biotinylated polystyrene microspheres, wereobtained from Spherotech Corporation. Other buffers and reagentsincluded phosphate-buffered saline (PBS) containing 0.5% BSA (sterilefiltered), and the streptavidin conjugated fluorophoresstreptavidin-Alexa 350, streptavidin-Alexa 488, and streptavidin-PE(phycoerythryn), each obtained from Molecular Probes. Binding reactionswere monitored with an inverted fluorescent microscope connected to avideo camera.

[0671] The analysis was conducted according to the following numberedsteps.

[0672] The fluid network of the chip was washed with water, then withPBS/BSA/Tween-20.

[0673] Beads were captured on the chip using its retention chamber.

[0674] Streptavidin-conjugates were loaded into reagent-wells on thechip (2 μL of each conjugate in 1 mL PBS).

[0675] The captured beads were exposed to each of the conjugates.

[0676] A 63× oil-immersion lens on the inverted microscope was used tomaximize fluorescent signal. Blue and green/red filter sets were used.

[0677] In some cases the rate of photobleaching by the detectionmechanism exceeded the rate at which fluorescent conjugates werecaptured by the beads. In these cases, the procedure was repeatedwithout constant exposure to V, opening the UV shutter only long enoughto document binding.

[0678] Results

[0679]FIG. 74 shows the results of portions of the analysis as selectedvideo frames during exposure of streptavidin-Alexa 488 conjugate toretained beads. In FIG. 74A, the beads have been loaded in chamber 270,but have not bound detectable amounts of the conjugate and are notdetectable. In FIG. 74B, beads 1550 are detectable above background. InFIG. 74C, they have become readily detectable, after unbound conjugateis washed out of the chamber. FIG. 74D shows beads 1550 under brightfield illumination to localize the beads and demonstrate that all beadsin the chamber are stained with conjugate.

[0680] Similar exposures to the other conjugates gave less intensestaining. Detectable staining with streptavidin-Alexa 350 was visible,but streptavidin-PE did not yield a detectable signal. However, moresensitive detection mechanisms, such as a laser scanning cytometer mayallow detection of streptavidin-PE binding.

Example 17 Measuring Ion Flux in Cells Using a Microfluidic System

[0681] This example describes analysis of intracellular ionconcentrations, such as calcium ion concentrations, using a microfluidicsystem; see FIG. 75.

[0682] Background

[0683] Calcium is a very important intracellular ion. It plays a vitalrole in the transduction of signals from the cell membrane to the cellcytoplasm and nucleus. A change in intracellular calcium levels is anindication that the cell is responding to a stimulus. Many stimuli causemobilization of calcium, either as an influx from the extracellularmedium or by release from intracellular pools. Fluorescent calciumindicators allow this mobilization to be observed.

[0684] Method

[0685] Materials used for measuring intracellular calcium levels were asfollows. A microfluidic chip was constructed based on a modified versionof system 850 of Example 7. Fluo 3/AM, a fluorescent Ca⁺² indicator dyewas obtained from Calbiochem, and used as a 5 mM stock. Ionomycin, freeacid form, was also obtained from Calbiochem. Cells were Jurkat T-cellsand were grown in RPMI media.

[0686] The analysis was conducted according to the following numberedsteps.

[0687] Cells were cultured in RPMI media.

[0688] Cells/media (5 mL) were pelleted at 1000 rpm for 5 min.

[0689] The cells were resuspended in RPMI containing 5 μM Fluo-3 (10 mLRPMI plus 8 μL FLUO-3 AM).

[0690] The cell/Fluo-3 mixture was incubated at 37° C. for 30 min toload the cells with indicator dye.

[0691] The cells were pelleted and washed twice with Hanks' balancedsalt solution (HBBS) containing 20 mM HEPES (200 μL 1M Hepes in 10 mLHBBS).

[0692] The cells were placed in the input reservoir of the chip.

[0693] The microscope and video camera were set up.

[0694] HBBS/Hepes buffer was pumped across cells, acting as a shieldbuffer to regulate exposure to reagent.

[0695] HBBS/Hepes containing ionomycin was pumped past the cells, but ina layer spaced from the cells by the shield buffer.

[0696] The flow of shield buffer flow was terminated, exposing the cellsto ionomycin.

[0697] Calcium flux was recorded with the video camera as ionomycincontacted the cells.

[0698] Results

[0699]FIG. 75 shows the results of the analysis, as selected videoframes, before and after exposure of Jurkat cells, loaded with indicatordye, to ionomycin. FIG. 75A shows two cells 1570 captured in retentionsite 1572 and visualized under bright field illumination. In FIG. 75B,these cells lack fluorescence before ionomycin exposure. In contrast,FIG. 75C reveals fluorescence (green signal) of cells 1570 very soonafter ionomycin exposure. A negative control demonstrated that ionomycinwas required for this fluorescence (not shown).

Example 18 Microfluidic Analysis of Cell-Surface Markers

[0700] This example describes a method for detection of cell-surfacemarkers, such as CD4 and CD8, on cultured T-cells using labeledantibodies.

BACKGROUND

[0701] The CD4 molecule recognizes an antigen that interacts with classII molecules of the major histocompatibility complex (MHC) and is theprimary receptor for the human immunodeficiency virus (HIV)(Dalgleish etal., 1984; Maddon et al., 1986). The cytoplasmic portion of the antigenis associated with the protein tyrosine kinase p56^(kk) (Rudd et al.,1989). The CD4 antigen may regulate the function of the CD3antigen/T-cell antigen receptor (TCR) complex (Kurrle et al., 1989). TheCD4 antibody reacts with monocytes/macrophages that have an antigendensity lower than that on helper/inducer T lymphocytes (Wood et al.,1983).

[0702] The CD8 antigen is present on the human suppressor/cytotoxicT-lymphocyte subset (Evans, et al., 1981; Ledbetter et al., 1981) aswell as on a subset of natural killer (NK) lymphocytes (Lanier et al.,1983). The CD8 antigenic determinant interacts with class I MHCmolecules, resulting in increased adhesion between the CD8⁺ Tlymphocytes and the target cells (Anderson et al., 1987; Eichmann etal., 1987; Gallagher et al., 1988). Binding of the CD8 antigen to classI MHC molecules enhances the activation of resting T lymphocytes. CD8recognizes an antigen expressed on the 32-kDa a-subunit of adisulfide-linked bimolecular complex (Moebius, 1989). The cytoplasmicdomain of the a-subunit of the CD8 antigen is associated with theprotein tyrosine kinase p56^(kk) (Rudd et al., 1989; Gallagher et al.,1989).

[0703] Determining the percentages of CD4+ and CD8+ lymphocytes may beuseful in monitoring the immune status of patients with immunedeficiency diseases, autoimmune diseases, or immune reactions. Therelative percentage of the CD4+ subset is depressed and the relativepercentage of the CD8+ subset is elevated in many patients withcongenital or acquired immune deficiencies such as severe combinedimmunodeficiency (SCID) and acquired immunodeficiency syndrome(AIDS)(Schmidt, 1989; Giorgi, 1990).

[0704] The percentage of suppressor/cytotoxic lymphocytes can be outsidethe normal reference range in some autoimmune diseases (Antel et al.,1986) and in certain immune reactions such as acute graft-versus-hostdisease (GVHD) and transplant rejection (Gratama et al., 1984; Bishop etal., 1986). The relative percentage of the CD8⁺ lymphocyte populationmay often be decreased in active systemic lupus erythematosus (SLE) butcan also be increased in SLE patients undergoing steroid therapy(Wolde-Mariam et al., 1984).

[0705] The CD4⁺/CD8⁺ (helper/suppressor) lymphocyte ratio, quantified asthe ratio of CD4 fluorescein isothiocyanate (FITC)-positive lymphocytesto CD8 phycoerythrin (PE)positive lymphocytes, has been used to evaluatethe immune status of patients with, or suspected of developing,autoimmune disorders or immune deficiencies (Antel et al., 1986;Wolde-Mariam et al., 1984; Smolen et al., 1982). In many cases, therelative percentages of helper lymphocytes decline and suppressorlymphocytes increase in immune deficiency states. These states may alsobe marked by T-cell lymphopenia (Ohno et al., 1988). In addition, theratio has been used to monitor bone marrow transplant patients for onsetof acute GVHD (Gratama et al., 1984).

[0706] The Jurkat cell, a human mature leukemic cell line,phenotypically resembles resting human T lymphocytes and has been widelyused to study T cell physiology. These cells are round, growing singlyor in clumps in suspension. They were established from a human T cellleukemia in the peripheral blood of a 14-year-old boy with acutelymphoblastic leukemia (ALL) at first relapse in 1976. This cell line isalso called “JM” (JURKAT and JM are derived from the same patient andare sister clones). Occasionally JM may be a subclone with somewhatdivergent features confirmed as human with IEF of AST, LDH, and NP.Jurkat cells have the following general restriction properties: CD2+,CD3+, CD4+, CD5+, CD6+, CD7+, CD8−, CD13−, CD19−, CD34+, TCRalpha/beta+,and TCRgamma/delta−.

[0707] Method

[0708] Materials used for analysis of CD4 and CD8 were as follows.Microfluidic chips was constructed based on a modified version of system850 of Example 7. Jurkat T-cells were cultured in RPMI.Fluorophore-conjugated antibodies, CD4-fluorescein isothiocyanate (FITC)and CD8-phycoerythryn (PE), were used. Buffer for dilution, focusing,washing, etc. was PBS containing 0.5% BSA. Data were collected with aninverted fluorescent microscope equipped with a video camera.

[0709] The analysis was conducted according to the following numberedsteps.

[0710] Jurkat cells were grown in RPMI and then pelleted (10 mL ofmedia/cells).

[0711] The cells were resuspended in 1 mL PBS containing 0.5% BSA.

[0712] Anti-CD4-FITC and anti-CD8-PE-antibody-conjugates were diluted1:100 in PBS containing 0.5% BSA.

[0713] The chip was prepared by running deionized water through themicrofluidic network and then was mounted on an inverted fluorescentmicroscope. The 100× or 63× oil-immersion lens was used to maximizefluorescent signal.

[0714] Cells were loaded onto the chip, positioned, and retained.

[0715] The diluted antibody-conjugates were loaded into separate reagentinput-wells of the chip.

[0716] Exposure to light from the UV lamp was minimized to avoidphotobleaching.

[0717] Anti-CD4-FITC was exposed to cells for 2 min.

[0718] The valve regulating CD4 antibody-conjugate flow was closed.

[0719] The shield-buffer flow line was opened to remove unboundantibodies.

[0720] The UV excitation shutter was opened and cell fluorescence wasrecorded.

[0721] When fluorescence was dim or invisible, the UV shutter was closedand steps 8 through 11 were repeated.

[0722] Step 12 was repeated until fluorescence was observed anddocumented.

[0723] As a negative control, steps 8 through 12 were repeated usinganti-CD8-PE.

[0724] Results

[0725] Anti-CD8 antibody-conjugate did not bind to Jurkat cells, andtherefore little or no red fluorescence was visible in the time frameneeded to visualize the green fluorescence of the anti-CD4antibody-conjugate. The procedure may be repeated with continuous UVexposure to observe antibody binding in real-time.

REFERENCES

[0726] Maddon P, Dalgleish A, McDougal J, Clapham P, Weiss R, Axel R.The T4 gene encodes the AIDS virus receptor and is expressed in theimmune system and the brain. Cell. 1986;47:333-348.

[0727] Dalgleish A, Beverly P, Clapham P, Crawford D, Greaves M, WeissR. The CD4 (T4) antigen is an essential component of the receptor forthe AIDS virus. Nature. 1984;312(December):763-767.

[0728] Rudd C, Burgess K, Barber E, Schlossman S. Monoclonal antibodiesto the CD4 and CD8 antigens precipitate variable amounts ofCD4/CD8-associated p56-lck activity. In: Knapp W, Dörken B, Gilks W R,et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens.Oxford: Oxford University Press; 1989:326-327.

[0729] Kurrle R. Cluster report: CD3. In: Knapp W, Dörken B, Gilks W R,et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens.Oxford: Oxford University Press; 1989:290-293.

[0730] Wood G, Warner N, Warnke R. Anti-Leu-3/T4 antibodies react withcells of monocyte/macrophage and Langerhans lineage. J Immunol.1983;131(1):212-216.

[0731] Evans R, Wall D, Platsoucas C, et al. Thymus-dependent membraneantigens in man: Inhibition of cell-mediated lympholysis by monoclonalantibodies to the TH₂ antigen. Proc Natl Acad Sci USA.1981;78(1):544-548.

[0732] Ledbetter J A, Evans R L, Lipinski M, Cunningham-Rundles C, GoodR A, Herzenberg L A. Evolutionary conservation of surface molecules thatdistinguish T lymphocyte helper/inducer and T cytotoxic/suppressorsubpopulations in mouse and man. J Exp Med. 1981;153(February):310-323.

[0733] Lanier L L, Le A M, Phillips J H, Warner N L, Babcock G F.Subpopulations of human natural killer cells defined by expression ofthe Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol. 1983; 131(4):1789-1796.

[0734] Anderson P, Blue M-L, Morimoto C, Schlossman S. Cross-linking ofT3 (CD3) with T4 (CD4) enhances the proliferation of resting Tlymphocytes. J Immunol. 1987;139:678-682.

[0735] Eichmann K, Johnson J, Falk I, Emmrich F. Effective activation ofresting mouse T lymphocytes by cross-linking submitogenic concentrationsof the T-cell antigen receptor with either Lyt-2 or L3T4. Eur J Immunol.1987;17:643-650.

[0736] Gallagher P, Fazekas de St. Groth B, Miller J. CD4 and CD8molecules can physically associate with the same T-cell receptor. ProcNatl Acad Sci USA. 1989;86: 10044-10048.

[0737] Moebius U. Cluster report: CD8. In: Knapp W, Dörken B, Gilks W R,et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens.Oxford: Oxford University Press;1989:342-343.

[0738] Bernard A, Boumsell L, Hill C. Joint report of the FirstInternational Workshop on Human Leucocyte Differentiation Antigens bythe investigators of the participating laboratories: T2 protocol. In:Bernard A, Boumsell L, Dausett J, Milstein C, Schlossman S, eds.Leucocyte Typing. Berlin: Springer-Verlag;1984:25-60.

[0739] Schmidt R. Monoclonal antibodies for diagnosis ofimmunodeficiencies. Blut. 1989;59:200-206.

[0740] Centers for Disease Control. Guidelines for the performance ofCD4⁺ T-cell determinations in persons with human immunodeficiency virusinfection. MMWR. 1992;41(No. RR-8):1-17.

[0741] Giorgi J, Hultin L. Lymphocyte subset alterations andimmunophenotyping by flow cytometry in HIV disease. Clin ImmunolNewslett. 1990;10(4):55-61.

[0742] Antel J, Bania M, Noronha A, Neely S. Defective suppressor cellfunction mediated by T8⁺ cell lines from patients with progressivemultiple sclerosis. J Immunol. 1986;137:3436-3439.

[0743] Gratama J, Naipal A, Oljans P, et al. T lymphocyte repopulationand differentiation after bone marrow transplantation: Early shifts inthe ratio between T4⁺ and T8⁺ T lymphocytes correlate with theoccurrence of acute graft-versus-host disease. Blood.1984;63(6):1416-1423.

[0744] Bishop G, Hall B, Duggin G, Horvath J, Sheil A, Tiller D.Immunopathology of renal allograft rejection analyzed with monoclonalantibodies to mononuclear cell markers. Kidney Internat.1986;29:708-717.

[0745] Wolde-Mariam W, Peter J. Recent diagnostic advances in cellularimmunology. Diagnost Med. 1984;7:25-32.

[0746] Smolen J, Chused T, Leiserson W, Reeves J, Alling D, Steinberg A.Heterogeneity of immunoregulatory T-cell subsets in systemic lupuserythematosus: Correlation with clinical features. Am J Med.1982;72:783-790.

[0747] Ohno T, Kanoh T, Suzuki T, et al. Comparative analysis oflymphocyte phenotypes between carriers of human immunodeficiency virus(HIV) and adult patients with primary immunodeficiency using two-colorimmunofluorescence flow cytometry. J Exp Med. 1988;154:157.

Example 19 Measuring Cell Lysis in a Microfluidic System

[0748] This example describes capture, lysis, and staining of cells.

[0749] Background

[0750] Acridine orange (AO) was used for staining. AO binds to singlestranded nucleic acids as a dimer, which fluoresces red in color, and todouble stranded nucleic acids as a monomer, which fluoresces green. Thisdifference in fluorescent wavelength is caused by differentialaccessibility of AO molecules to the nucleic acid binding sites. AOfluorescence is also pH sensitive, staining acidic organelles, such aslysosomes, orange.

[0751] Method

[0752] Materials used for measuring lysis were as follows. Microfluidicchips was constructed based on system 250 of Example 2. Jurkat T-cellswere cultured in RPMI. Acridine Orange was dissolved at 5 μg/ml in PBS.Solutions or liquids to lyse cells included PBS containing 0.05%hydrogen peroxide, deionized water, PBS containing 2% TWEEN 20 (0.2 μmfiltered), and WINDEX. Data were collected on an inverted fluorescentmicroscope equipped with a video camera.

[0753] The analysis was conducted according to the following numberedsteps.

[0754] Jurkat cells were grown in RPMI and pelleted (10 mL of culturemedia/cells).

[0755] The cells were resuspended in 5 mL PBS containing 5 μg/mlAcridine Orange, or left unstained for use on a control chip. For thecontrol chip, proceed to step 5.

[0756] The cells were incubated 10 min at room temperature.

[0757] The cells were pelleted and washed twice in PBS.

[0758] The cells were resuspended in 1 mL PBS.

[0759] The chip was preparing by washing the microfluidic network withdeionized water, and then was mounted on an inverted fluorescentmicroscope. The microscope's 63× oil-immersion lens was used to maximizefluorescent signal.

[0760] The cells were loaded onto the chip, positioned, and retained.

[0761] PBS containing peroxide was loaded into a reagent-well of thechip.

[0762] Exposure of the chip to light from the UV lamp was minimized, tominimize photobleaching.

[0763] The UV shutter was opened to expose stained cells to fluorescentlight.

[0764] PBS containing peroxide was pumped over the cells for 2 min oruntil lysis or photobleaching occurred.

[0765] Cells were then exposed sequentially to PBS/2% TWEEN-20, WINDEX,and finally water.

[0766] Results

[0767] The conditions of peroxide, TWEEN, and WINDEX did not lyse thecells on the first attempt of this experiment. Subsequently, water wasused successfully to demonstrate cell lysis. Lysis probably occurredunder the other conditions, but was not as obvious. Jurkat cells arefairly robust and may not be a good model cell line for this experiment.

Example 20 Inducing and Detecting Cell Apoptosis in a MicrofluidicEnvironment

[0768] This example describes induction and detection of cell apoptosisin a microfluidic system; see FIG. 76.

[0769] Background

[0770] Apoptosis, also termed programmed cell death, is a carefullyregulated process of cell death that occurs as a normal part ofdevelopment. Inappropriately regulated apoptosis is implicated indisease states, such as Alzheimer's disease and cancer. Apoptosis isdistinguished from necrosis, or accidental cell death, by characteristicmorphological and biochemical changes, including compaction andfragmentation of the nuclear chromatin, shrinkage of the cytoplasm, andloss of membrane asymmetry.¹⁻⁵

[0771] Phosphatidylserine (PS) distribution also can act as a marker forapoptosis. In normal viable cells, phosphatidylserine is located on thecytoplasmic side of the cell membrane. However, in apoptotic cells, PSis translocated from the inner to the outer leaflet of the plasmamembrane, thus exposing PS to the cell exterior.⁶ In leukocyteapoptosis, PS on the outer surface of the cell marks the cell forrecognition and phagocytosis by macrophages.^(7,8) The humananticoagulant, annexin V, is a 35-36 kD Ca⁺²-dependentphospholipid-binding protein that has a high affinity for PS.⁹ Annexin Vcan identify apoptotic cells by binding to PS exposed on the outerleaflet.¹⁰ Bound annexin V may be detected through a dye, a specificbinding member conjugated to annexin V, an anti-annexin-V antibody,and/or the like.

[0772] Hydrogen peroxide has been shown to induce markers of apoptosis,such as PS translocation, in cultured cells. The cellular toxicity ofhydrogen peroxide (H₂O₂) is initiated by oxidative stress, resulting inrapid modification of cytoplasmic constituents, depletion ofintracellular glutathione (GSH) and ATP, a decrease in NAD⁺ level, anincrease in free cytosolic Ca²⁺, and lipid peroxidation.¹¹ H₂O₂ alsoactivates the mitochondria permeability transition pore and the releaseof cytochrome c.¹² In the cytoplasm, cytochrome c, in combination withApaf-1, activates caspase-9, leading to the activation of caspase-3 andsubsequent apoptosis¹³⁻¹⁵.

[0773] Method

[0774] This example demonstrates induction and detection of cellapoptosis in a microfluidic system. Jurkat cells are positioned andretained in a microfluidic system, and then programmed cell death isinitiated by exposure of these cells to hydrogen peroxide. Translocationof PS to the outer membrane leaflet is monitored with annexin V, tomeasure apoptosis. At the same time, cells are exposed to propidiumiodide, which stains cells with disrupted membranes, an indicator ofnecrosis rather than apoptosis.

[0775] Materials used were as follows. Microfluidic chips wereconstructed based on system 250 of Example 2. Jurkat T-cells werecultured in RPMI. The VYBRANT Apoptosis Assay Kit #2 was obtained fromMolecular Probes, Eugene, Oreg. This kit includes fluorophore-conjugatedannexin V (green) and propidium iodide (red). Data were collected on aninverted fluorescent microscope equipped with a video camera.

[0776] The analysis was conducted according to the following numberedsteps.

[0777] The video camera was turned on.

[0778] Cells were trapped in the retention chamber of the chip.

[0779] Annexin-V-conjugate was loaded into reagent well #1 of the chip.

[0780] Propidium iodide was loaded into reagent well #2 of the chip.

[0781] Binding Buffer (BB) was loaded into the shield buffer well of thechip.

[0782] The cells were perfused with BB for 5 min.

[0783] The cells were perfused with annexin-V-conjugate for 5 min.

[0784] Cells were checked for staining. (Note: This is a negativecontrol. No staining occurred at this stage because the cells had notapoptosed.)

[0785] The valves regulating flow of the shield buffer and reagent wellswere each closed.

[0786] The BB was replaced with 800 μM H₂O₂ in PBS.

[0787] The cells were exposed to the H₂O₂/PBS by opening the valveregulating flow from of the shield buffer.

[0788] Cells were observed under light microscopy during induction ofapoptosis.

[0789] After 15 min, the valve regulating flow of the shield buffer wasclosed. The well was washed with BB, and then replaced with BB.

[0790] The cells were then perfused with BB for 5 min.

[0791] The valve for the annexin-V-conjugate was opened, and theshielding buffer valve was closed.

[0792] The cells were exposed to the annexin-V-conjugate for 5 min.

[0793] The valve controlling the annexin-V-conjuagate was closed, andthe BB valve was opened to wash the cells.

[0794] The cells were exposed to excitation light by opening themicroscope shutter. Green fluorescence indicated a positive reaction forphosphatidylserine.

[0795] The valve that regulates flow of propidium iodide (“the P1valve”) was opened, while the valve that regulates BB (“the BB valve”)was closed.

[0796] After 2 min, the BB valve was reopened, and the P1 valve wasclosed.

[0797] After washing for 5 min, the fluorescent shutter was opened whileusing the red filter set on the microscope.

[0798] Finally, the BB was replaced with water, and the cells were lysedand then re-exposed to the P1.

[0799] Results

[0800]FIG. 76 shows selected video frames from this analysis. In panelA, cells 1590 have been trapped in chamber 270 and are visible underbright field illumination. Panels B and C compare labeling of cells withthe annexin-V-conjugate before (B) and after (C) exposure to hydrogenperoxide. Cells 1590 do not label with the annexin-conjugate beforeexposure to hydrogen peroxide (panel B), but a weak annexin-conjugatesignal is detectable after hydrogen peroxide exposure (panel C),demonstrating that at least some of the cells have initiated apoptosis.Panels D-F compare propidium iodide staining of cells 1590 at differenttimes during the analysis. Panels D and E show no propidium iodidestaining, either before or after induction of apoptosis by exposure tohydrogen peroxide. In contrast, panel F reveals detectablepropidium-iodide staining after exposure of cells to water, whichrenders the cells necrotic.

REFERENCES

[0801] Immunol. Cell Biol. 76, 1 (1998).

[0802] Cytometry 27, 1 (1997).

[0803] J. Pharmacol Toxicol. Methods 37, 215 (1997).

[0804] FASEB J. 9, 1277 (1995).

[0805] Am J. Pathol. 146, 3 (1995).

[0806] Cytometry 31, 1 (1998).

[0807] J. Immunol. 148, 2207 (1992).

[0808] J. Immunol. 151, 4274 (1993).

[0809] J. Biol. Chem. 265, 4923 (1990).

[0810] Blood 84, 1415 (1994).

[0811] Am. J. Physiol. 273, G7 (1997).

[0812] Free Radic. Biol. Med. 24, 624 (1998).

[0813] FEBS Lett. 447, 274 (1999).

[0814] Cell 91, 479 (1997).

[0815] Annu. Rev. Cell Dev. Biol. 15, 269 (1999).

Example 21 Analysis of Aquatic Microorganisms in a Microfluidic System

[0816] This example describes the capture and visualization of aquaticmicroorganisms, such as plankton, using a microfluidic system.

[0817] Background

[0818] Plankton are a very diverse group of marine and fresh waterorganisms that spend some or all of their lives drifting in water.Plankton represent both the animal and plant kingdoms and include arange of sizes from submicron to over a centimeter. These seeminglylistless organisms play critical roles, both positive and negative, inthe health of not only other aquatic organisms but also in thecomposition of the earth's atmosphere. For example, these organisms arethought to produce a large fraction of the earth's oxygen. In addition,they play a critical role in global carbon dioxide exchange, removingmuch of the excess carbon dioxide produced by burning fossil fuels andsending this carbon dioxide to the ocean floor. In contrast, someplankton are infamous for their negative impact on the economy. Forexample, explosive population growth of dinoflagellate plankton producea toxic “red tide” that poisons fish and shellfish. However, occurrencesof red tides are difficult to predict and/or prevent, resulting inextensive fish-kills and beach closures, which have a large economicimpact. Therefore, systems are needed to manipulate, treat, and analyzeplankton, including laboratory or natural populations that benefit orharm the environment.

[0819] Method and Results

[0820] This example provides a microfluidic system capable ofmanipulating and detecting small plankton, particularly picoplankton(0-2 μm), ultraplankton (2-5 μm), and/or nannoplankton (5-60 μm).Plankton may be retained, treated, and/or detected in an integratedmicrofluidic environment.

[0821] Plankton were manipulated and detected in a microfluidic systemas follows. A sample of seawater was collected from San Francisco Bayand centrifuged to concentrate organisms in the sample. A 20 μL aliquotof the concentrated sample was loaded into the input reservoir ofmicrofluidic system 250, described in Example 2 above.Naturally-fluorescent plankton were retained in chamber 270 and detectedsuccessfully by fluorescent microscopy (not shown).

[0822] This method of this example may be modified by changing anysuitable parameters. For example, plankton may be collected fromfreshwater sources or cultured, an aqueous plankton sample may be loadeddirectly into a microfluidic environment without concentration, and/orretained plankton may be exposed to any suitable reagents.Alternatively, or in addition, microfluidic systems may be used thatsort a heterogeneous population of plankton according to a physicalproperty (such as size or density, among others) or a measuredproperty/characteristic (such as labeling with a dye and/or specificbinding member).

Example 22 Analysis of Membrane Trafficking in a Microfluidic Systemusing Membrane Dyes

[0823] This example describes microfluidic analysis of membranetrafficking pathways in cells treated with membrane-labeling dyes.

[0824] Background

[0825] Studies of vesicle trafficking often rely on optically detectabledyes that label membranes. Brief exposure of cells to such a dye resultsin labeling of the surface-membrane of these cells. Subsequent dyemovement to interior membranes, such as endosomes, Golgi apparatuses,lysosomes, and/or endoplasmic reticulum, tracks corresponding transit ofsurface membranes, receptors, and/or ligands, among others, throughintracellular vesicle trafficking pathways. Using this approach, cellendocytic, recycling, degradative, and/or secretory pathways may bemonitored and analyzed.

[0826] Some “FM” dyes available from Molecular Probes bind to cellmembranes. Thus these FM membrane dyes may be used as general-purposeprobes for endocytosis, because they are generally nontoxic. FM membranedyes are virtually non-fluorescent in aqueous solution, but becomeintensely fluorescent upon association with a membrane.

[0827] Goals and Method

[0828] The goals of this analysis included the following. I) Define thestaining conditions for two FM membrane dyes, FM 1-43 and FM 4-64, usingJurkat cells. FIGS. 77 and 78 show the structure and excitation/emissionspectra of these dyes. These two FM dyes have substantiallynonoverlapping emission spectra. II) Test the affinity of FM dyes formicrofluidic chips formed with PDMS, to define a background level ofstaining. III) Trap a Jurkat cell in a microfluidic chip and performtwo-color staining of the cell using the two FM membrane dyes.

[0829] Materials used for this analysis included the following. FM 1-43and FM 4-64 were obtained from Molecular Probes. Microfluidic chips wereproduced based on system 250 of Example 2. Results were collected andrecording using an inverted fluorescent microscope equipped with a videocamera.

[0830] Conditions for labeling Jurkat cells with FM membrane dyes weredetermined with the following labeling protocol.

[0831] Cultured Jurkat cells (5 mL of cells/media) were pelleted bycentrifugation at 1000 rpm for 5 min.

[0832] The cell pellet was washed twice with PBS.

[0833] The cell pellet was resuspended in 2 mL PBS.

[0834] Aliquots (500 μL) of the resulting cell suspension were dispensedinto four microcentrifuge tubes.

[0835] Dye was added to each of the four tubes as follows: no dye wasadded to tube #1, FM 1-43 was added to tube #2, FM 4-64 was added totube #3, and both FM 1-43 and FM 64 were added to tube #4. The final dyeconcentration for each dye was 2 μM.

[0836] The cells were observed with the fluorescent microscope.

[0837] Each staining condition was documented by saving digital imagefiles.

[0838] Labeling of the microfluidic chip with the FM membrane dyes todetermine background signal was carried out as follows.

[0839] Each dye was diluted to a final concentration of 2 μM in PBS.

[0840] FM 1-43 (5 μL) was introduced into a first chip.

[0841] FM 4-64 (5 μL) was introduced into a second available chip.

[0842] A mixture of the FM 1-43 and 4-64 dyes (1:1) was introduced intoa third chip.

[0843] Each dye-loaded chip was observed using a fluorescent microscope.

[0844] The level of background staining was determined relative tofluorescence intensity of the cells stained with FM dyes in part Aabove.

[0845] Cells were labeled with FM dyes in a microfluidic system asfollows.

[0846] Unlabeled Jurkat cells were loaded and captured in a microfluidicchip using PBS as a carrier buffer.

[0847] Each FM membrane dye (5 μL) was placed in one of the two reagentwells on the chip.

[0848] Chip features and cells were visualized using minimalincandescent light.

[0849] The video camera was turned on, and the 100× oil-immersionobjective on the fluorescent scope was used.

[0850] The first FM membrane dye (1-43) was delivered to the cells.

[0851] The fluorescent signal was observed.

[0852] The second FM membrane dye (4-64) was delivered to the cells.

[0853] The fluorescent signal was observed.

[0854] Steps 5-8 were repeated as necessary until the signal intensitywas maximized.

[0855] Results

[0856] The results of the three protocols are as follows.

[0857] Protocol A produced significant labeling of Jurkat cells with thedyes after a 5 minute incubation at room temperature. Each dye stainedthe cells with sufficient intensity to visualize using the fluorescentmicroscope. For example, FIG. 79 shows Jurkat cells stained with FM1-43. However, the emission profile of each dye was not distinguishableas a discrete color using the green/red filter set on a Leicamicroscope. Properly selected filter sets may allow a two-color assayusing these dyes.

[0858] Protocol B produced significant background labeling ofmicrofluidic chips formed with PDMS, using either dye. The PDMS may besurface-modified to minimize binding of these dyes to the chip.

[0859] Protocol C was foiled by the high background produced by dyebinding to PDMS. After trapping a single cell in the chip, FM 1-43 boundto the chip more efficiently than to the membrane of the trapped cell.

Example 23 Capturing Cells in Single-Cell or Multi-Cell MicrofluidicChambers

[0860] This example describes capture of a single cell or a cellpopulation in a microfluidic system; see FIGS. 80-82.

[0861]FIG. 80 shows a single cell captured at a retention site using achip fabricated generally according to system 850 of Example 7. In FIG.80A, cells 1610 follow a divided flow path extending in oppositedirections above retention site 1612. In FIG. 80B, a trapped cell 1614is positioned at the retention site.

[0862] Multiple cells were captured in a larger retention chamber formedby a chip fabricated generally according to system 250 of Example 2.FIGS. 81A, 81B, and 81C show empty chamber 270, the chamber with twocells, and with six cells, respectively. FIG. 82 shows a similar captureof cells, but here the cells are prelabeled with a fluorescent dye sothat the cells are easily visible as bright green using fluorescentmicroscopy. FIGS. 82A and 82B show a chamber with only three cells andduring the entry of a fourth cell, respectively.

Example 24 Fixing and Staining Cells in a Microfluidic System

[0863] This example describes the use of a microfluidic system to fix acell with an organic solvent, methanol, and label the cell with acridineorange; see FIG. 83.

[0864] All cell manipulations and treatments were as described inExample 2. FIG. 83A shows a single cell 1630 retained at the bottom ofretention site 1632. The cell is barely visible due to the low level oflight used. The cell was perfused with methanol to fix the cell, andvisible cell-shrinkage was evident (not shown). FIG. 83B shows that thecell exhibits no fluorescence. However, after the cell was perfused witha solution of acridine orange, the cell fluoresces brightly (see FIG.83C).

Example 25 Microfluidic Mechanism for Measuring Cell Secretion

[0865] This example describes the structure and use of a softlithography-based, microfluidic system for measuring secretion ofmolecules, complexes, and/or small particles from cells.

[0866] Many cell analyses measure release, and/or secretion of materialsfrom cells. In some cases, the cells secrete material naturally. Forexample, neurons are analyzed for their ability to secreteneurotransmitters at neural synapses; endocrine cells for secretion ofendocrine hormones, such as insulin, growth hormone, prolactin, steroidhormones, etc.; and a broad range of cell types for secretion ofcytokines. In other cases, cells are lysed to define an aspect of theirinternal contents. However, in any of these cases, a secreted orreleased material of interest may no longer be held in a fixed positionby the cells, and thus may be free to diffuse into the ambient solution.Accordingly, such secreted or released materials may be difficult toanalyze without concentrating them and/or without using immobilized,high-affinity binding partners, for example, in ELISA.

[0867] Microfluidic systems may ameliorate some of the difficultiesassociated with measuring material released from cells, but mayintroduce additional considerations. In microfluidic systems, cells maybe grown in isolated chambers having small volumes, as described abovein Example 10. The chambers may maintain released materials in the smallvolumes, promoting subsequent analysis. However, to maintain thereleased materials in a concentrated form, the chambers may be isolatedfrom other portions of the microfluidic network. Such isolated chambersdo not promote ready analysis of the released materials, since thematerials may be isolated from analytical reagents and may be difficultto collect without substantially diluting the released materials.Therefore, a microfluidic mechanism is needed that allows materialreleased from cells to be collected and/or analyzed in a distinctfluidic compartment that is not part of a primary fluidic layer of amicrofluidic system.

[0868] This example provides a microfluidic system having a cell chamberand a separate material collection compartment that communicatefluidically through a semi-permeable membrane. The semi-permeablemembrane permits movement of material that is secreted/released fromcells, but prevents movement of cells themselves. The membrane may beform a portion of a fluid layer, or interface with a fluid layer aboveand/or below the fluid layer. When disposed below, the membrane may formsome or all of the substrate for the fluid layer. Accordingly,secreted/released material may pass through the membrane for collectionand/or analysis in another compartment of the fluid layer, a compartmentabove the fluid layer, and/or below the substrate. For example, themicrofluidic system may include a layer similar to the base layer ofExample 11.

Example 26 Microfluidic Analysis of a Heterogeneous ParticlePopulation—Part II

[0869] This example describes microfluidic systems for sorting andanalyzing heterogeneous populations of particles, such as blood samples,based on differences in particle size; see FIGS. 84-88. Example 26expands upon aspects of Example 15 above.

[0870] Description

[0871] This example provides a microfluidic system 1650 that selectivelyretains and analyzes larger particles from a mixture of larger andsmaller particles; see FIGS. 84 and 85. System 1650 includes an inputmechanism 1652, a positioning mechanism 1654, a filtration mechanism1656, a retention mechanism 1658, a perfusion mechanism 1660, a releasemechanism 1662, and a flow-based detection mechanism 1664, among others.These mechanisms may be grouped into a first set for inputting sampleand size-selecting the sample, and a second set for retaining, treating,measuring, and outputting the size-selected sample.

[0872] The first set of mechanisms may functionally interconnect asfollows. Input mechanism 1652 introduces particles from a particlesample placed in particle input-reservoir 1666, into microfluidicnetwork 1668 of system 1650. Particles are moved by positioningmechanism 1654 to filtration mechanism 1656 by flow along inlet channel1670. Filtration mechanism 1656 may act as a size-dependent andregulatable retention mechanism, or prefilter, that removes smallerparticles from the inputted particles, while retaining larger particles.After suitable filtration, the larger particles may be released fromfiltration mechanism 1656 and moved by positioning mechanism 1654 towardretention mechanism 1658.

[0873] The second set of mechanisms may functionally interconnect asfollows. Positioning mechanism 1654 may use a first focusing mechanism1672 to focus and direct particles toward retention mechanism 1658.Particles retained by retention mechanism 1658 may be perfused withdesired reagents from perfusion mechanism 1660, then released by releasemechanism 1662. Released cells may be moved by positioning mechanism1654 toward flow-based detection mechanism 1664. During positioning,cells may be focused into a single stream of particles by a secondfocusing mechanism 1674. Finally, detected cells may be passed to outputmechanism 1676.

[0874] System 1650 may include a plurality of regulators, or valves,that may regulate various aspects of the mechanisms described above; seeFIG. 85. Valve V1 may regulate input mechanism 1652. Valve V2 mayregulate alternative input mechanism 1678. Alternative input mechanism1678 may provide an alternative source of input fluid, and may be usedto supply particle-free fluid for washing filtration mechanism 1658, forcarrying particles from filtration mechanism to first focusing mechanism1672 and on to retention mechanism 1658, and/or the like. Valve V3 mayregulate input from first reagent reservoir 1680. Valve V4 may regulateinput from second reagent reservoir 1682. Valve V5 may regulate flow ofa shield buffer to space reagents from retained particles until thedesired moment for beginning treatment. V6 may regulate flow through afirst waste channel 1684. V7 may regulate release mechanism 1662. V8 mayregulate flow through a second waste channel 1686. V9 may regulate flowtoward detection mechanism 1664. Finally, V10 may regulate afilter-release mechanism 1688 that regulates release of particles fromregulatable retention mechanism 1656.

[0875] Further aspects of input mechanism 1652, positioning mechanism1654, retention mechanism 1658, perfusion mechanism 1660, releasemechanism 1662, and output mechanism 1676 elsewhere in Section XIII.

[0876] Applications

[0877] The description that follows exemplifies use of system 1650 forseparation and analysis of white blood cells from a sample of wholeblood. However, system 1650 may be suitable for use with anyheterogeneous (or homogeneous) population of particles.

[0878] System 1650 first separates white blood cells from smaller redblood cells and platelets. These separated white blood cells aredirected to a retention site, retained, and then processed by theperfusion mechanism to stain the retained white blood cells. Thesestained cells are then released from the retention site and thenpositioned to a separate flow-based detection site. The detection sitethen detects a characteristic of the stained cells, based on thestaining method/reagents used.

[0879] A chip fabricated according to system 1650 may be readied for useas follows. First, the chip may be loaded with water. Next, when all thechannels are filled, the water may be replaced with a buffer solution.At this point, the following valves generally are closed: V1, V2, V3,V4, V5, V9, and V10. By contrast, the following valves generally areopen: V6, V7, and V8. All input reservoirs may be loaded with theirrespective buffers/reagents. However, particle input-reservoir 1666typically is not loaded yet. Each waste reservoir 1692, 1694, 1696, and1698 may be emptied (or is already empty).

[0880] A sample of whole blood may be loaded and filtered as follows. Analiquot of blood is loaded into particle input-reservoir 1666. Valve V1may be opened and the blood allowed to flow into filtration mechanism1656. FIG. 86 shows the operation of filtration mechanism 1656 ingreater detail. A first set of particle-selective channels 1700, forexample, channels that are about 7 μm wide and 5 μm high, may bedisposed along the walls of inlet channel 1702. A second set ofparticle-selective channels or chamber channels 1704 also may bedisposed around the perimeter of capture chamber 1706. Accordingly, redblood cells may travel to flow-through chambers 1708 and then wastereservoirs 1692, 1694, along a substantial area formed by inlet channel1702 and chamber 1706. In particular, travel of red blood cells throughparticle-selective channels 1700 from inlet channel 1702 may avoidclogging chamber channels 1704. However, the white blood cells may beretained in chamber 1704, because they cannot pass through channels 1700and may not travel past chamber 1704 because filter-release mechanism1688 (valve V10) is closed.

[0881] White blood cells retained in capture chamber 1706 may be washedas follows. After a suitable number of white blood cells have enteredchamber 1706, valve V1 may be closed so that no more whole blood entersinlet channel 1702 and chamber 1706. Then, valve V2 may be opened toallow the carrying buffer provided by alternative input mechanism 1678to wash residual red blood cells out of chamber 1706. At this point,waste reservoirs 1692, 1694 may be emptied to avoid reverse flow of thered blood cells back into chamber 1706.

[0882] Filtered white blood cells may be retained by retention mechanism1658 as follows; see FIGS. 85-87. Valve V10 may be opened to allow thefiltered white blood cells from chamber 1706 to be released. Thereleased cells may be focused by first focusing mechanism 1672 andcarried toward retention site 1710 (see FIG. 87). Flow of carryingbuffer from alternative input mechanism 1678 may act during this processto reposition the white blood cells from chamber 1706 to retention site1710.

[0883] Retained white blood cells may be stained with reagents asfollows. Valve V10 may be closed to prevent additional white blood cellsfrom leaving chamber 1706 and entering retention site 1710. Next, valveV6 may be closed to facilitate directing reagents along a flow pathtoward the retained white blood cells by perfusion mechanism 1660. Next,white blood cells may be stained or otherwise treated/processed usingperfusion mechanism 1660, as described elsewhere in Section XIII,particularly Example 2. Pump P1 may be used by perfusion mechanism 1660to actively move reagents, buffer, and/or fluid during particletreatment (see FIG. 85). At this point, the valves may be in thefollowing configuration. Valves V1, V3, V4, V5, V6, V9, are V10 closed.Valves V2, V7, and V8 are open. After cell treatment has been completed,pump P1 may be turned off, and valves V3, V4, and V5 may be closed toterminate action of perfusion mechanism 1660.

[0884] Treated/processed cells may be released and detected as follows;see FIGS. 84, 85, 87, and 88. Pump P2 may be turned on. This pump may beused to pull fluid, particles, and/or reaction products toward detectionmechanism 1664 and waste (output) reservoir 1698. Next, valve V8 may beclosed and valve V9 opened (see FIG. 87). With this valve configuration,fluid and particle may be directed toward waste reservoir 1698 insteadof waste reservoir 1696 (see FIG. 85). At this point, each focusingreservoir 1712, 1714, 1716, 1718 may be refilled with buffer and wastereservoir 1698 may be emptied. Then, partial or complete closure ofvalve V7 may be used to release white blood cells from retentionmechanism 1658. During release, buffer flowing from reservoirs 1712,1714, or alternative input mechanism 1678, may be used to carry thereleased white blood cells toward detection mechanism 1664. Bufferflowing from reservoirs 1716, 1718 may act in second focusing mechanism1674, to position (focus) the released cells to a desiredcross-sectional portion of outlet channel 1720, generally a centralportion (see FIG. 88). After cell focusing, outlet channel 1720 mayconstrict to a narrowed channel 1722, which may facilitate positioningthe cells in single file, that is, one-by-one at detection site 1724,rather than in groups.

[0885] System 1650 may be used to measure any suitable aspect of a bloodsample or other inputted particle population, including samples frompatients, research subjects, volunteers, forensic studies, cadavers,etc. Suitable aspects may include analysis of leukemias, anemias, bloodabnormalities, blood health, genetic diseases, infections, ratios ofspecific blood cell types, presence of nonblood cells, and/or the like.Exemplary leukemias may include acute lymphoblastic leukemias, chronicmyelogenous leukemias, acute myelogenous leukemias, acute lymphoidleukemias, chronic lymphocystic leukemias, and/or juvenilemyelolymphocystic leukemias, among others. Exemplary anemias and/orgenetic diseases may include aplastic anemias, Faconi anemias,sickle-cell anemias, and/or the like. Other aspects or characteristicsof blood cells (or other heterogeneous particle populations) that may besuitable for analysis are described above in Sections VIII and XII.

[0886]FIG. 89 is a top plan view of a perfusion device for exposingparticles to an array of different reagents or different reagentconcentrations. Here, microfluidic passage device 2000 provides aplurality of growth/perfusion chambers 2030 for loading particles, suchas cells, through loading passage 2010 which is controlled by valvingline 2020 which is in operable communication with control input 2070,and which, when actuated, isolates each chamber 2030 from one another.Particles may then be flushed out the chambers 2030 by opening valvingline 2020 and pushing fluid from loading passage 2010 through eachchamber 2030 towards exit passage 2080. Once each chamber 2030 is loadedwith particles, such as cells, and isolated, valve line 2040, which isin operable communication with control input 2140, then opens to permitflow of reagent and diluent, such as media or a fluid that dilutes thereagent, through flow lines 2120, which originate from a diluentreservoir 2110, and optionally, reagent reservoir 2100, which may hold areagent for exposure to the particles. The ratio of diluent to reagentmay be controlled by valving, or, preferably, by controlling the bore ofthe lines connecting the diluent reservoir 2110 to flow line 2120 andreagent reservoir 2100 to flow line 2120. Diluent and reagent are thenfed into chambers 2030 by pumping action caused by, for example, aperistaltic pump 2090, which is actuated by pump input lines 2150 a-c,thus particles are perfused with reagent/diluent. Diluent, in the caseof cells, may be cell culture media. Effluent from chambers 2030 may becollected into waste reservoir 2050.

[0887]FIGS. 90 through 94 depict a top plan view of a device being usedto measure the response of cells to a chemo-attractant. Microfluidicpassage device 2200 provides reagent loading chamber 2230, whereinreagent is metered into reagent chamber 2300 by the opening of valve2210 and blind filling reagent into reagent chamber 2300. Once reagentchamber 2230 is filled, particles 2320, such as cells, which werepreviously introduced into particle chamber 2300 are then exposed to agradient of reagent upon the opening of valve 2220, valve 2210,preferably, remains closed during the formation of the gradient. FIG. 91shows reagent entering into gradient forming mechanism 2250, which haschannels 2270 for limiting reagent flow into particle chamber 2320. FIG.92 depicts the advancement of reagent towards particle chamber 2300.FIGS. 93 and 94 depict the movement of particles 2320 toward channels2270 where the chemo-attractant reagent is emanating from.

[0888]FIG. 95 is a close-up top plan view of a perfusion chamber withassociated valving system. Particles, such as cells, can be loaded intoa series of particle chambers 2450 by opening isolation valve line 2430which, when closed, isolates each chamber 2450 from each other.Particles do not enter flow line 2460 since they are retained in chamber2450 by screen or comb 2490, which each obstruction is spaced-apart fromthe other at a distance less than that of the particle, so as to retainthe particle on one side of the screen or comb 2490. In use, particlesare introduced into chamber 2450 by the opening of isolation valve line2430 which allows the particles to flow through and fill each chamber2450. Once filled with the desired amount of particles, isolation valves2430 are closed to isolate each chamber 2450 from each other, and thenflow valves 2440 are opened to allow for flow of reagent through chamber2450 to perfuse the particles with reagent. Once an experiment iscomplete, flow valves 2440 may then be closed, isolation valves 2430 maythan be opened to flush out particles. If the particles are adherentcells, such cells can be liberated if attached by exposing such adheredcells to a cell dislodging reagent such as trypsin. Once liberated, thecells can be flushed out of the system, and the system reused.

[0889] The disclosure set forth above may encompass one or more distinctinventions, with independent utility. Each of these inventions has beendisclosed in its preferred form(s). These preferred forms, including thespecific embodiments thereof as disclosed and illustrated herein, arenot intended to be considered in a limiting sense, because numerousvariations are possible. The subject matter of the inventions includesall novel and nonobvious combinations and subcombinations of the variouselements, features, functions, and/or properties disclosed herein.

What is claimed is:
 1. A microfluidic device for treating a particlecomprising: (a) an input mechanism for introducing a fluid samplecontaining a particle; (b) a microfluidic passage in fluid communicationwith said input mechanism; (c) a positioning mechanism in fluidcommunication with said microfluidic passage, said positioning mechanismfor positioning said particle in said microfluidic passage whilecontained in said fluid sample; (d) a retention mechanism for retainingsaid particle upon being positioned by said positioning means; (e) atreatment mechanism in communication with said retention mechanism forselectively treating said particle to produce a treatment response whilebeing retained within said retention mechanism; and, (f) a measurementmechanism for measuring said treatment response, if any, of saidparticle.
 2. The microfluidic device of claim 1 further comprising arelease mechanism for releasing said particle from said retentionmechanism.
 3. The microfluidic device of claim 2 further comprising anoutput mechanism for outputting said particle from said microfluidicdevice.
 4. The microfluidic device of claim 2 further comprising a cellculture mechanism for culturing said particle.
 5. The microfluidicdevice of claim 1 further comprising a control mechanism for determiningaspects of the flow rate or path of the sample fluid or other fluid. 6.The microfluidic device of claim 5, wherein said control mechanism is avalve in communication with said microfluidic passage.
 7. Themicrofluidic device of claim 6, wherein said microfluidic device isformed from a multi-layer elastomeric block and, wherein said valve isformed from an elastomeric membrane within said elastomeric block. 8.The microfluidic device of claim 6, wherein said control mechanism is apump in communication with said microfluidic passage.
 9. Themicrofluidic device of claim 8, wherein said microfluidic device isformed from a multi-layer elastomeric block and, wherein said pump isformed from an elastomeric membrane within said elastomeric block. 10.The microfluidic device of claim 1, wherein said microfluidic devicecomprises a multi-layered elastomeric block having a control layerhaving an elastomeric membrane deflectable into said microfluidicpassage in a fluidic layer to determine the flow rate or path of a fluidin said microfluidic passage.
 11. The microfluidic device of claim 1,wherein said microfluidic device comprises a layer including a materialselected from the group consisting of elastomers, polydimethylsiloxane,plastic, polystyrene, polypropylene, polycarbonate, glass, ceramic,silicon, sol-gels, metal, metalloids, metal oxides, biological polymers,mixtures thereof, particles, proteins, gelatins, polylysine, serumalbumin, collagen, nucleic acids, and microoganisms.
 12. Themicrofluidic device of claim 1, wherein said microfluidic passage has isless than about 500 micrometers wide.
 13. The microfluidic device ofclaim 1, wherein said microfluidic passage further comprises an adjacentpassage joining said microfluidic passage at a junction or branch, saidadjacent passage being selected from the group consisting of inletpassage, outlet passage, particle passage, reagent passage, and wastepassage.
 14. The microfluidic device of claim 13, wherein said adjacentpassage is a dead-end passage.
 15. The microfluidic device of claim 13further comprising said adjacent passage manipulating said particle. 16.The microfluidic device of claim 15, wherein said particle manipulatingis selected from the group of positioning, sorting, retaining, treating,detecting, propagating, storing, mixing, and releasing.
 17. Themicrofluidic device of claim 1, wherein said particle is selected fromthe group consisting of cells, eukaryotic cells, prokaryotic cells,plant cells, animal cells, hybridoma cells, bacterial cells, yeastcells, viruses, organelles, beads, and vesicles.
 18. The microfluidicdevice of claim 17, wherein said particle is a plurality or an aggregateof particles.
 19. The microfluidic device of claim 18, wherein saidplurality of particles is a complex mixture containing differentparticles.
 20. The microfluidic device of claim 19, wherein said complexmixture containing different particles is whole blood or serum or bodilyfluid.
 21. The microfluidic device of claim 1, wherein said particle isan egg or embryo.
 22. The microfluidic device of claim 1, wherein theinput mechanism is a receptacle or well in fluid communication with saidmicrofluidic passage.
 23. The microfluidic device of claim 22, whereinthe input mechanism has a volume greater than a volume defined by saidmicrofluidic passage.
 24. The microfluidic device of claim 1 furthercomprising a facilitating mechanism in communication with or acting uponsaid input mechanism.
 25. The microfluidic device of claim 24, whereinsaid facilitating mechanism is selected from the group consisting ofgravity, fluid pressure, centrifugal pressure, pump pressure, andnegative fluid pressure.
 26. The microfluidic device of claim 1, whereinsaid positioning mechanism is a direct positioning mechanism or anindirect positioning mechanism.
 27. The microfluidic device of claim 26,wherein said direct positioning mechanism is a force selected from thegroup consisting of optical, electrical, magnetic, and gravitational.28. The microfluidic device of claim 27, wherein said electrical forceis selected from the group consisting of electrophoretic,electroosmotic, electroendoosmotic, and dielectrophoretic.
 29. Themicrofluidic device of claim 26, wherein said indirect positioningmechanism is a longitudinal indirect positioning mechanism or atransverse indirect positioning mechanism.
 30. The microfluidic deviceof claim 29, wherein said indirect positioning mechanism is facilitatedby a pump or a valve associated with said microfluidic device.
 31. Themicrofluidic device of claim 29, wherein said transverse indirectpositioning mechanism is facilitated by a fluid flow stream at a passagejunction, laterally disposed region of reduced fluid flow, or channelbend.
 32. The microfluidic device of claim 31, wherein said passagejunction is unifying or dividing.
 33. The microfluidic device of claim29, wherein said transverse indirect positioning mechanism is a laminarflow-based transverse positioning means.
 34. The microfluidic device ofclaim 29, wherein said transverse indirect positioning mechanism is astochastic transverse positioning mechanism.
 35. The microfluidic deviceof claim 34, wherein said stochastic transverse positioning mechanismrandomly selects said particle from a population of particles by lateralseparation of said particle in said sample fluid from a main flow regionto a reduced flow region.
 36. The microfluidic device of claim 29,wherein said transverse indirect positioning mechanism is a centrifugalforced-based transverse positioning mechanism.
 37. The microfluidicdevice of claim 1 wherein said retention mechanism selectively retainssaid particle at a pre-selected region within said microfluidic device.38. The microfluidic device of claim 37, wherein said retentionmechanism retains said particle by overcoming or counteracting a forcecaused by said positioning mechanism.
 39. The microfluidic device ofclaim 1, wherein said retention mechanism is a trap or barrier-basedretention mechanism.
 40. The microfluidic device of claim 39, whereinsaid barrier-based retention mechanism is restricts longitudinalmovement of said particle in or adjacent said microfluidic passage. 41.The microfluidic device of claim 38, wherein said retention mechanism isa protrusion extending, fixedly or transiently, into or adjacent saidmicrofluidic passage to restrict longitudinal movement of said particle.42. The microfluidic device of claim 26, wherein said direct positioningmechanism is a chemical retention mechanism.
 43. The microfluidic deviceof claim 42, wherein said chemical retention mechanism is based on aspecific affinity between said particle and said retention mechanism.44. The microfluidic device of claim 1, wherein said treatment mechanismis a fluid-mediated mechanism or a non-fluid mediated mechanism.
 45. Themicrofluidic device of claim 1, wherein said treatment mechanism exposessaid particle to a reagent or physical condition.
 46. The microfluidicdevice of claim 45, wherein said reagent is selected from the groupconsisting of chemical modulator, biological modulator, agonist,antagonist, hormone, ligand, small molecule, peptide, protein,carbohydrate, lipid, receptor, nutrient, toxin, drug, chemicalsubstance, compound, ion, polymer, nucleic acid, material, complex,mixture, aggregate, dye, stain, fluorescent dye, detection agent, assayagent, substrate, substrate inhibitor, antibody, labeled substance, andbiological particle.
 47. The microfluidic device of claim 46, whereinsaid reagent attracts or repels said particles.
 48. The microfluidicdevice of claim 45, wherein said reagent induces or inhibits cellparticle proliferation.
 49. The microfluidic device of claim 45, whereinsaid reagent is cytotoxic.
 50. The microfluidic device of claim 44,wherein said fluid-mediated mechanism further comprises a fluidtreatment and wherein said particles are introduced to said fluidtreatment.
 51. The microfluidic device of claim 44, wherein saidfluid-mediated mechanism functions in conjunction with the functioningof said positioning mechanism.
 52. The microfluidic device of claim 51,wherein said positioning mechanism is a transverse positioning mechanismfor moving said particle into and out of said fluid-mediated mechanismto modulate exposure of said particle to said treatment fluid.
 53. Themicrofluidic device of claim 45, wherein said physical condition isselected from the group consisting of heat, light, radiation, sub-atomicparticles, electric fields, magnetic fields, pressure, acousticalpressure, gravity, and micro-gravity.
 54. The microfluidic device ofclaim 1, wherein said measurement mechanism is a detector associatedwith said microfluidic device that detects a characteristic of saidparticle or caused by said particle.
 55. The microfluidic device ofclaim 54, wherein said detector is selected from the group consisting ofspectroscopes, electronic sensors, hydrodynamic sensors, imagingsystems, and photon detectors.
 56. The microfluidic device of claim 54,wherein said detector detects multiple values.
 57. The microfluidicdevice of claim 54, wherein said detector employs a detection mode thatis selected from the group consisting of time-independent,time-dependent, and averaged.
 58. The microfluidic device of claim 54,wherein said detector is a spectroscopic detector that detects a signalproduced of a type selected from the group consisting of absorption,luminescence, photoluminescence, chemiluminescence, electroluminescence,magnetic resonance, nuclear resonance, electron spin resonance,scattering, electron scattering, light scattering, neutron scattering,diffraction, circular dichroism, optical rotation, fluorescenceintensity, fluorescence resonance energy transfer, fluorescencepolarization, fluorescence lifetime, total internal reflectionfluorescence, fluorescence correlation spectroscopy, fluorescencerecovery after photobleaching, fluorescence activated cell sorting, andphosphorescent.
 59. The microfluidic device of claim 54, wherein saiddetector is an electrical detector capable of detecting a signalselected from the group consisting of current, voltage, resistance,capacitance, and power.
 60. The microfluidic device of claim 54, whereinsaid detector is a hydrodynamic detector which detects a hydrodynamicinteraction between said particle and a fluid, another particle, or saidmicrofluidic passage.
 61. The microfluidic device of claim 60, whereinsaid interaction included a hydrodynamic interaction selected from thegroup consisting of chromatography, sedimentation, viscometry,electrophoresis.
 62. The microfluidic device of claim 54, wherein saiddetector is an imaging detector for creating and analyzing images ofsaid particle(s).
 63. The microfluidic device of claim 54, wherein saiddetector detects a biological response produced by said particle(s). 64.The microfluidic device of claim 63, wherein said biological response isselected from the group consisting of chemotaxis, biotaxis, senescence,apoptosis, proliferation, differentiation, morphological change, pHchange, and calcium uptake.
 65. The microfluidic device of claim 1,further comprising a detection site, wherein said particle or product ofsaid particle, is detected by said detector.
 66. The microfluidic deviceof claim 65, wherein said detection site is within said microfluidicdevice.
 67. The microfluidic device of claim 65, wherein said detectionsite is located external to said microfluidic device.
 68. Themicrofluidic device of claim 54, wherein said detector detects acharacteristic of said particle, directly or indirectly, saidcharacteristic being selected from the group consisting of particleidentity, particle number, particle concentration, composition,structure, sequence, activity, molecular character, morphology,phenotype, genotype, growth, apoptosis, necrosis, lysis, alive/deadratio, position in cell cycle, activity of signal pathway,differentiation, transcriptional activity, substrate attachment,cell-cell interaction, translational activity, replication activity,transformation, heat shock response, motility, spreading, membraneintegrity, chemotaxis, and neurite outgrowth.
 69. The microfluidicdevice of claim 2, wherein said release mechanism operates by removing aretaining force caused by said retaining mechanism.
 70. The microfluidicdevice of claim 2, wherein said release mechanism operates by overcominga retaining force caused by said retaining mechanism.
 71. Themicrofluidic device of claim 2, wherein said release mechanism operatesby rendering ineffective a retaining force caused by said retainingmechanism.
 72. The microfluidic device of claim 2, further comprisingdirecting said particle to another region within or external saidmicrofluidic device.
 73. The microfluidic device of claim 72, whereinsaid another region is selected from the group consisting of a secondpositioning mechanism, a second detection mechanism, a second retentionmechanism, and an output mechanism.
 74. The microfluidic device of claim73, wherein said second retention mechanism is a cell culture chamber.75. The microfluidic device of claim 3, further comprising said outputmechanism outputting said particle to a location selected from the groupconsisting of an internal sink, and external sink, a waste site, acollection site, a cell growth chamber, and an external cell cultureplate.
 76. A method for perfusing cells with a reagent comprising thesteps of: (a) providing a microfluidic device having (i) a cell growthchamber, a cell inlet in communication with said chamber, said cellinlet having an in valve in operable communication therewith to valvefluid flow through said cell inlet into said chamber, wherein said cellscan pass through said cell inlet into said chamber when said inlet valveis open, but cannot pass through said cell inlet when said inlet valveis closed; and, (ii) a reagent inlet for inputting said reagent intosaid chamber, said reagent inlet having a reagent valve in operablecommunication with said reagent inlet for valving fluid flow throughsaid reagent into said chamber, said inlet or said chamber having anretention mechanism for retaining said cells in said chamber whilepermitting flow of said reagent into said chamber when said reagentvalve is open; wherein when said cells are loaded into said chamber, andsaid cell valve is closed, said cells are retained in said chamber whilesaid reagent valve is open and closed; (b) opening said cell inlet valveand introducing said cells into said chamber; (c) closing said cellinlet valve; (d) opening said reagent valve to introduce said reagentinto said chamber; and, (e) introducing said reagent into said chamberwhile retaining said cells inside of said chamber thereby perfusing saidcells with said reagent.
 77. A method for treating a particle comprisingthe steps of: (i) providing a microfluidic device comprising: (a) aninput mechanism for introducing a fluid sample containing a particle;(b) a microfluidic passage in fluid communication with said inputmechanism; (c) a positioning mechanism in fluid communication with saidmicrofluidic passage, said positioning mechanism for positioning saidparticle in said microfluidic passage while contained in said fluidsample; (d) a retention mechanism for retaining said particle upon beingpositioned by said positioning means; (e) a treatment mechanism incommunication with said retention mechanism for selectively treatingsaid particle to produce a treatment response while being retainedwithin said retention mechanism; and, (f) a measurement mechanism formeasuring said treatment response, if any, of said particle. (ii)introducing said sample fluid containing said particle into said inputmechanism; (iii) positioning said particle with said positioningmechanism so that said particle is retainable by said retentionmechanism; (iv) retaining said particle with said retaining mechanism;(v) exposing said particle to said treatment by said treatmentmechanism; (vi) measuring said treatment response caused directly orindirectly by said particle upon exposure to said treatment.
 78. Themethod of claim 77 wherein said microfluidic device further comprises arelease mechanism for releasing said particle from said retentionmechanism, and said method further comprises the step of releasing saidparticle from said retaining mechanism.
 79. The method of claim 78,wherein said microfluidic device further comprises an output mechanismfor outputting said particle from said microfluidic device, and saidmethod further comprises the step of outputting said particle from saidmicrofluidic device by said output mechanism.
 80. The method of claim78, wherein said microfluidic device further comprises a cell culturemechanism for culturing said particle, and the method further comprisesthe step of culturing said particle in said cell culture mechanism. 81.The method of claim 77, wherein said microfluidic device furthercomprises a control mechanism for determining aspects of the flow rateor path of the sample fluid or other fluid, and the method furthercomprises the step of determining the flow rate or path of the samplefluid or other fluid by said control mechanism.
 82. The method of claim81, wherein said control mechanism is a valve in communication with saidmicrofluidic passage, and the method further comprises valving saidsample fluid or other fluid with said valve.
 83. The microfluidic deviceof claim 82, wherein said microfluidic device is formed from amulti-layer elastomeric block and, wherein said valve is formed from anelastomeric membrane within said elastomeric block, and wherein saidvalving occurs by deflecting said elastomeric membrane into saidmicrofluidic passage.
 84. The method of claim 82, wherein said controlmechanism is a pump in communication with said microfluidic passage, andwherein said determining the flow rate or path of said sample fluidoccurs by actuation of said pump.
 85. The method of claim 84, whereinsaid microfluidic device is formed from a multi-layer elastomeric blockand, wherein said pump is formed from an elastomeric membrane withinsaid elastomeric block, and wherein said pump is actuated by deflectinga series of elastomeric membranes into said microfluidic passage in aselected sequence.
 86. The method of claim 77, wherein said microfluidicdevice comprises a multi-layered elastomeric block having a controllayer having an elastomeric membrane deflectable into said microfluidicpassage in a fluidic layer to selectively determine the flow rate orpath of a fluid in said microfluidic passage.
 87. The method of claim77, wherein said microfluidic passage further comprises an adjacentpassage joining said microfluidic passage at a junction or branch, saidadjacent passage being selected from the group consisting of inletpassage, outlet passage, particle passage, reagent passage, and wastepassage, and said method further comprises the step of selectivelydetermining the path of said particle to said adjacent passage.
 88. Themethod of claim 87, wherein said adjacent passage is a dead-end passage,and wherein said selectively determining includes introducing saidsample fluid into said dead-end passage wherein said sample fluiddisplaces gas, if present, in said dead-end passage to fill saiddead-end passage with said sample fluid.
 89. The method of claim 87further comprising said adjacent passage manipulating said particle. 90.The method of claim 89, wherein said particle manipulating includesretaining said particle in addition to either positioning, sorting,treating, detecting, propagating, storing, mixing, or releasing saidparticle.
 91. The method of claim 77, wherein said particle is selectedfrom the group consisting of cells, eukaryotic cells, prokaryotic cells,plant cells, animal cells, hybridoma cells, bacterial cells, yeastcells, viruses, organelles, beads, and vesicles, and wherein saidtreating step treats said particle.
 92. The method of claim 91, whereinsaid particle is a plurality or an aggregate of particles, and saidmethod further comprises a sorting step to sort out and separate orisolate a desired particle from said plurality of particles, and saidtreating step treats said separated or isolated particle.
 93. The methodof claim 92, wherein said plurality of particles is a complex mixturecontaining different particles, and said sorting step sorts out at leastone type of particle from other different particles in said complexmixture.
 94. The method of claim 93, wherein said complex mixturecontaining different particles is whole blood or serum or bodily fluid,and said sorting step selects for at least one type of cell from thewhole blood or serum.
 95. The method of claim 77, wherein said particleis an egg or embryo, and said treatment is a step towards in-vitrofertilizing or manipulating said egg or embryo, respectively.
 96. Themethod of claim 77, wherein the input mechanism is a receptacle or wellin fluid communication with said microfluidic passage, and said methodfurther comprises the step of introducing said fluid sample into saidreceptacle.